Methods and products for expressing proteins in cells

ABSTRACT

The present invention relates in part to nucleic acids encoding proteins, therapeutics comprising nucleic acids encoding proteins, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods and products for altering the DNA sequence of a cell are described, as are methods and products for inducing cells to express proteins using synthetic RNA molecules. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.

PRIORITY

The present application is a continuation of International ApplicationNo. PCT/US13/68118, filed on Nov. 1, 2013, which claims priority to U.S.Provisional Application No. 61/721,302, filed on Nov. 1, 2012, U.S.Provisional Application No. 61/785,404, filed on Mar. 14, 2013, and U.S.Provisional Application No. 61/842,874, filed on Jul. 3, 2013, thecontents of which are herein incorporated by reference in theirentireties. The present application is related to U.S. application Ser.No. 13/465,490, filed on May 7, 2012, International Application No.PCT/US2012/067966, filed on Dec. 5, 2012, and U.S. application Ser. No.13/931,251, filed on Jun. 28, 2013, the contents of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates in part to nucleic acids encodingproteins, therapeutics comprising nucleic acids encoding proteins,methods for inducing cells to express proteins using nucleic acids,methods, kits and devices for transfecting, gene editing, andreprogramming cells, and cells, organisms, and therapeutics producedusing these methods, kits, and devices.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:FAB_(—)005_SeqList_ST25.txt; date recorded: Apr. 30, 2015; file size:255 KB).

BACKGROUND Synthetic RNA and RNA Therapeutics

Ribonucleic acid (RNA) is ubiquitous in both prokaryotic and eukaryoticcells, where it encodes genetic information in the form of messengerRNA, binds and transports amino acids in the form of transfer RNA,assembles amino acids into proteins in the form of ribosomal RNA, andperforms numerous other functions including gene expression regulationin the forms of microRNA and long non-coding RNA. RNA can be producedsynthetically by methods including direct chemical synthesis and invitro transcription, and can be administered to patients for therapeuticuse.

Cell Reprogramming and Cell-Based Therapies

Cells can be reprogrammed by exposing them to specific extracellularcues and/or by ectopic expression of specific proteins, microRNAs, etc.While several reprogramming methods have been previously described, mostthat rely on ectopic expression require the introduction of exogenousDNA, which can carry mutation risks. DNA-free reprogramming methodsbased on direct delivery of reprogramming proteins have been reported.However, these methods are too inefficient and unreliable for commercialuse. In addition, RNA-based reprogramming methods have been described(See, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010.5,107; Warren et al. Cell Stem Cell. 2010. 7,618-630; Angel. MIT Thesis.2011. 1-89; and Lee et al. Cell. 2012. 151,547-558; the contents of allof which are hereby incorporated by reference). However, existingRNA-based reprogramming methods are slow, unreliable, and inefficientwhen performed on adult cells, require many transfections (resulting insignificant expense and opportunity for error), can reprogram only alimited number of cell types, can reprogram cells to only a limitednumber of cell types, require the use of immunosuppressants, and requirethe use of multiple human-derived components, including blood-derivedHSA and human fibroblast feeders. The many drawbacks of previouslydisclosed RNA-based reprogramming methods make them undesirable for bothresearch and therapeutic use.

Gene Editing

Several naturally occurring proteins contain DNA-binding domains thatcan recognize specific DNA sequences, for example, zinc fingers (ZFs)and transcription activator-like effectors (TALEs). Fusion proteinscontaining one or more of these DNA-binding domains and the cleavagedomain of Fold endonuclease can be used to create a double-strand breakin a desired region of DNA in a cell (See, e.g., US Patent Appl. Pub.No. US 2012/0064620, US Patent Appl. Pub. No. US 2011/0239315, U.S. Pat.No. 8,470,973, US Patent Appl. Pub. No. US 2013/0217119, U.S. Pat. No.8,420,782, US Patent Appl. Pub. No. US 2011/0301073, US Patent Appl.Pub. No. US 2011/0145940, U.S. Pat. No. 8,450,471, U.S. Pat. No.8,440,431, U.S. Pat. No. 8,440,432, and US Patent Appl. Pub. No.2013/0122581, the contents of all of which are hereby incorporated byreference). However, current methods for gene editing cells areinefficient and carry a risk of uncontrolled mutagenesis, making themundesirable for both research and therapeutic use. Methods for DNA-freegene editing of somatic cells have not been previously explored, norhave methods for simultaneous or sequential gene editing andreprogramming of somatic cells. In addition, methods for directly geneediting cells in patients (i.e., in vivo) have not been previouslyexplored, and the development of such methods has been limited by a lackof acceptable targets, inefficient delivery, inefficient expression ofthe gene-editing protein/proteins, inefficient gene editing by theexpressed gene-editing protein/proteins, due in part to poor binding ofDNA-binding domains, excessive off-target effects, due in part tonon-directed dimerization of the FokI cleavage domain and poorspecificity of DNA-binding domains, and other factors. Finally, the useof gene editing in anti-bacterial, anti-viral, and anti-cancertreatments has not been previously explored.

Accordingly, there remains a need for improved compositions and methodsfor the expression of proteins in cells.

SUMMARY OF THE INVENTION

The present invention provides, in part, compositions, methods,articles, and devices for inducing cells to express proteins, methods,articles, and devices for producing these compositions, methods,articles, and devices, and compositions and articles, including cells,organisms, and therapeutics, produced using these compositions, methods,articles, and devices. Unlike previously reported methods, certainembodiments of the present invention do not involve exposing cells toexogenous DNA or to allogeneic or animal-derived materials, makingproducts produced according to the methods of the present inventionuseful for therapeutic applications.

In some aspects, synthetic RNA molecules with low toxicity and hightranslation efficiency are provided. In one aspect, a cell-culturemedium for high-efficiency transfection, reprogramming, and gene editingof cells is provided. Other aspects pertain to methods for producingsynthetic RNA molecules encoding reprogramming proteins. Still furtheraspects pertain to methods for producing synthetic RNA moleculesencoding gene-editing proteins.

In one aspect, the invention provides high-efficiency gene-editingproteins comprising engineered nuclease cleavage domains. In anotheraspect, the invention provides high-fidelity gene-editing proteinscomprising engineered nuclease cleavage domains. Other aspects relate tohigh-efficiency gene-editing proteins comprising engineered DNA-bindingdomains. Still further aspects pertain to high-fidelity gene-editingproteins comprising engineered DNA-binding domains. Still furtheraspects relate to gene-editing proteins comprising engineered repeatsequences. Some aspects relate to methods for altering the DNA sequenceof a cell by transfecting the cell with or inducing the cell to expressa gene-editing protein. Other aspects relate to methods for altering theDNA sequence of a cell that is present in an in vitro culture. Stillfurther aspects relate to methods for altering the DNA sequence of acell that is present in vivo.

In some aspects, the invention provides methods for treating cancercomprising administering to a patient a therapeutically effective amountof a gene-editing protein or a nucleic-acid encoding a gene-editingprotein. In one aspect, the gene-editing protein is capable of alteringthe DNA sequence of a cancer associated gene. In another aspect, thecancer-associated gene is the BIRC5 gene. Still other aspects relate totherapeutics comprising nucleic acids and/or cells and methods of usingtherapeutics comprising nucleic acids and/or cells for the treatment of,for example, type 1 diabetes, heart disease, including ischemic anddilated cardiomyopathy, macular degeneration, Parkinson's disease,cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severecombined immunodeficiency, hereditary sensory neuropathy, xerodermapigmentosum, Huntington's disease, muscular dystrophy, amyotrophiclateral sclerosis, Alzheimer's disease, cancer, and infectious diseasesincluding hepatitis and HIV/AIDS. In some aspects, the nucleic acidscomprise synthetic RNA. In other aspects, the nucleic acids aredelivered to cells using a virus. In some aspects, the virus is areplication-competent virus. In other aspects, the virus is areplication-incompetent virus.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1A depicts RNA encoding the indicated proteins and containingadenosine, 50% guanosine, 50% 7-deazaguanosine, 70% uridine, 30%5-methyluridine, and 5-methylcytidine, resolved on a denaturingformaldehyde-agarose gel.

FIG. 1B depicts RNA encoding the indicated proteins and containingadenosine, 50% guanosine, 50% 7-deazaguanosine, 50% uridine, 50%5-methyluridine, and 5-methylcytidine, resolved on a denaturingformaldehyde-agarose gel.

FIG. 2 depicts primary human neonatal fibroblasts reprogrammed by fivetransfections with RNA encoding reprogramming proteins. Cells were fixedand stained for Oct4 protein. Nuclei were counterstained with Hoechst33342.

FIG. 3A depicts primary human adult fibroblasts.

FIG. 3B depicts the primary human adult fibroblasts shown in FIG. 3A,reprogrammed by seven transfections with RNA encoding reprogrammingproteins. Arrows indicate colonies of reprogrammed cells.

FIG. 3C depicts a large colony of reprogrammed primary human adultfibroblasts.

FIG. 4A depicts the location of a TALEN pair targeting the human CCR5gene. Single-lines indicate the TALEN binding sites. Double-linesindicate the location of the 432 mutation.

FIG. 4B depicts synthetic RNA encoding the TALEN pair of FIG. 4A,resolved on a denaturing formaldehyde-agarose gel.

FIG. 4C depicts the results of a SURVEYOR assay testing thefunctionality of the RNA of FIG. 4B on human dermal fibroblasts(GM00609). The appearance of the 760 bp and 200 bp bands in the samplegenerated from cells transfected with RNA indicates successful geneediting. The percentage below each lane indicates the efficiency of geneediting (percentage of edited alleles).

FIG. 4D depicts a line-profile graph of the “Neg” and “TALENs” lanes ofFIG. 4C. Numbers indicate the integrated intensity of the three bands,relative to the total integrated intensity.

FIG. 4E depicts the results of a SURVEYOR assay performed as in FIG. 4C,and also including a sample generated from cells that were transfectedtwice with RNA (the lane labeled “2x”).

FIG. 4F depicts simultaneous gene editing and reprogramming of primaryhuman cells (GM00609) using synthetic RNA. Images show representativecolonies of reprogrammed cells.

FIG. 4G depicts the results of direct sequencing of the CCR5 gene ingene-edited, reprogrammed cells generated as in FIG. 4F. Four of thenine lines tested contained a deletion between the TALEN binding sites,indicating efficient gene editing.

FIG. 5 depicts the results of a SURVEYOR assay performed as in FIG. 4C,except using RNA targeting the human MYC gene, and containing eithercanonical nucleotides (“A, G, U, C”) or non-canonical nucleotides (“A, 7dG, 5 mU, 5 mC”). The dark bands at 470 bp and 500 bp indicatehigh-efficiency gene editing.

FIG. 6 depicts the results of a SURVEYOR assay performed as in FIG. 4C,except using RNA targeting the human BIRC5 gene, and containing eithercanonical nucleotides (“A, G, U, C”) or non-canonical nucleotides (“A, 7dG, 5 mU, 5 mC”). The dark band at 710 bp indicates high-efficiency geneediting.

FIG. 7A depicts HeLa cells (cervical carcinoma) transfected with RNAtargeting the human BIRC5 gene (RiboSlice). Cells were transfected witheither a single RNA (“2x Survivin L”) or equal amounts of each member ofan RNA pair (“Survivin L+R”), with the same total amount of RNAdelivered in each case. As shown in the right panel, cells transfectedwith the RNA pair became enlarged, and exhibited fragmented nuclei andmarkedly reduced proliferation, demonstrating the potent anti-canceractivity of RiboSlice.

FIG. 7B depicts HeLa cells transfected with RNA targeting the humanBIRC5 gene as in FIG. 7A. Cells were subsequently fixed and stained forsurvivin protein. Nuclei were counterstained with Hoechst 33342. Thelarge, fragmented nuclei of cells transfected with RiboSlice areindicated with arrows.

FIG. 8 depicts primary human adult fibroblasts reprogrammed usingsynthetic RNA. Arrows indicate compact colonies of cells that exhibit amorphology indicative of reprogramming.

FIG. 9 depicts synthetic RNA encoding the indicated gene-editingproteins, resolved on a denaturing formaldehyde-agarose gel.

FIG. 10A depicts the results of a SURVEYOR assay testing theeffectiveness of the RNA of FIG. 9 on human dermal fibroblasts. Cellswere lysed approximately 48 h after transfection. Bands corresponding todigestion products resulting from successful gene editing are indicatedwith asterisks. Lane labels are of the form “X.Y”, where X refers to theexon from which DNA was amplified, and Y refers to the gene-editingprotein pair. For example, “1.1” refers to the gene-editing protein pairtargeting the region of exon 1 closest to the start codon. “X.N” refersto untransfected cells.

FIG. 10B depicts the results of a SURVEYOR assay testing the toxicity ofthe RNA of FIG. 9 on human dermal fibroblasts. Cells were lysed 11 daysafter transfection. Lanes and bands are labeled as in FIG. 10A. Theappearance of the bands indicated with asterisks demonstrates that thetransfected cells retained high viability.

FIG. 11 depicts the results of a study designed to test the safety ofRNA encoding gene-editing proteins in vivo. The graph shows the meanbody weight of four groups of mice (10 animals in each group), includingone untreated group, one vehicle-only group, one group treated withRiboSlice via intratumoral injection, and one group treated withRiboSlice via intravenous injection. For all treated groups, animalswere given 5 doses, every other day, from day 1 to day 9 Animals werefollowed until day 17. The lack of a statistically significantdifference between the mean body weights of the four groups demonstratesthe in vivo safety of RiboSlice.

FIG. 12A depicts the results of a SURVEYOR assay testing theeffectiveness of gene-editing proteins comprising various 36amino-acid-long repeat sequences. Human dermal fibroblasts were lysedapproximately 48 h after transfection with RNA encoding gene-editingproteins containing the indicated repeat sequence. The bandcorresponding to the digestion product resulting from successful geneediting is indicated with an asterisk. Lane labels refer to the aminoacids at the C-terminus of the repeat sequence. “Neg.” refers tountransfected cells.

FIG. 12B depicts the results of a SURVEYOR assay testing theeffectiveness of gene-editing proteins in which every other repeatsequence is 36 amino acids long. Human dermal fibroblasts were lysedapproximately 48 h after transfection with RNA encoding gene-editingproteins containing the indicated repeat sequence. The bandcorresponding to the digestion product resulting from successful geneediting is indicated with an asterisk. Lane labels refer to the aminoacids at the C-terminus of the repeat sequences. “Neg.” refers tountransfected cells.

FIG. 13A depicts the results of a study designed to test the safety andefficacy of RiboSlice AAV replication-incompetent virus carrying nucleicacids encoding gene-editing proteins in vivo. The graph shows the meanbody weight of three groups of mice carrying subcutaneous tumorscomprising human glioma cells, including one untreated group (notreatment control, “NTC”, n=6), one group treated with AAV encoding GFP(“GFP”, n=2) via intratumoral injection, and one group treated withRiboSlice AAV encoding gene-editing proteins targeting the BIRC5 gene(“RiboSlice”, n=2) via intratumoral injection Animals were dosed on day1 for the GFP group, and days 1 and 15 for the RiboSlice group. Animalswere followed until day 25. The lack of a statistically significantdifference between the mean body weights of the three groupsdemonstrates the in vivo safety of RiboSlice AAV.

FIG. 13B depicts the normalized tumor volumes of the animals in thestudy shown in FIG. 13A. The slower increase in normalized tumor volumein the group treated with RiboSlice AAV compared to both the NTC and GFPgroups demonstrates the in vivo efficacy of RiboSlice AAV.

FIG. 14 depicts the results of a SURVEYOR assay testing theeffectiveness of gene-editing proteins, as in FIG. 12B. “RiboSlice”refers to gene-editing proteins in which every other repeat sequence is36 amino acids long. “w.t.” refers to untransfected cells.

FIG. 15 depicts RNA encoding the indicated proteins and containingadenosine, 50% guanosine, 50% 7-deazaguanosine, 60% uridine, 40%5-methyluridine, and 5-methylcytidine, resolved on a denaturingformaldehyde-agarose gel.

FIG. 16 depicts the results of an assay testing the integration of arepair template into the APP gene. The appearance of the 562 bp and 385bp bands in the sample generated from cells transfected with RNA and arepair template indicates successful integration of a PstI restrictionsite. “−” refers to an undigested sample, “+” refers to a sample treatedwith PstI restriction nuclease.

DEFINITIONS

By “molecule” is meant a molecular entity (molecule, ion, complex,etc.).

By “RNA molecule” is meant a molecule that comprises RNA.

By “synthetic RNA molecule” is meant an RNA molecule that is producedoutside of a cell or that is produced inside of a cell usingbioengineering, by way of non-limiting example, an RNA molecule that isproduced in an in vitro-transcription reaction, an RNA molecule that isproduced by direct chemical synthesis or an RNA molecule that isproduced in a genetically-engineered E. coli cell.

By “transfection” is meant contacting a cell with a molecule, whereinthe molecule is internalized by the cell.

By “upon transfection” is meant during or after transfection.

By “transfection reagent” is meant a substance or mixture of substancesthat associates with a molecule and facilitates the delivery of themolecule to and/or internalization of the molecule by a cell, by way ofnon-limiting example, a cationic lipid, a charged polymer or acell-penetrating peptide.

By “reagent-based transfection” is meant transfection using atransfection reagent.

By “cell-culture medium” is meant a medium that can be used for cellculture, by way of non-limiting example, Dulbecco's Modified Eagle'sMedium (DMEM) or DMEM+10% fetal bovine serum (FBS).

By “complexation medium” is meant a medium to which a transfectionreagent and a molecule to be transfected are added and in which thetransfection reagent associates with the molecule to be transfected.

By “transfection medium” is meant a medium that can be used fortransfection, by way of non-limiting example, Dulbecco's ModifiedEagle's Medium (DMEM) or DMEM/F12.

By “recombinant protein” is meant a protein or peptide that is notproduced in animals or humans. Non-limiting examples include humantransferrin that is produced in bacteria, human fibronectin that isproduced in an in vitro culture of mouse cells, and human serum albuminthat is produced in a rice plant.

By “lipid carrier” is meant a substance that can increase the solubilityof a lipid or lipid-soluble molecule in an aqueous solution, by way ofnon-limiting example, human serum albumin or methyl-beta-cyclodextrin.

By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Oct4protein (SEQ ID NO: 8), mouse Oct4 protein, Oct1 protein, a proteinencoded by POU5F1 pseudogene 2, a DNA-binding domain of Oct4 protein oran Oct4-GFP fusion protein. In some embodiments the Oct4 proteincomprises an amino acid sequence that has at least 70% identity with SEQID NO: 8, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95%identity with SEQ ID NO: 8. In some embodiments, the Oct4 proteincomprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 8. Or in other embodiments, the Oct4 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 8.

By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Sox2protein (SEQ ID NO: 9), mouse Sox2 protein, a DNA-binding domain of Sox2protein or a Sox2-GFP fusion protein. In some embodiments the Sox2protein comprises an amino acid sequence that has at least 70% identitywith SEQ ID NO: 9, or in other embodiments, at least 75%, 80%, 85%, 90%,or 95% identity with SEQ ID NO: 9. In some embodiments, the Sox2 proteincomprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 9. Or in other embodiments, the Sox2 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 9.

By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human Klf4protein (SEQ ID NO: 10), mouse Klf4 protein, a DNA-binding domain ofKlf4 protein or a Klf4-GFP fusion protein. In some embodiments the Klf4protein comprises an amino acid sequence that has at least 70% identitywith SEQ ID NO: 10, or in other embodiments, at least 75%, 80%, 85%,90%, or 95% identity with SEQ ID NO: 10. In some embodiments, the Klf4protein comprises an amino acid sequence having from 1 to 20 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 10. Or in other embodiments, the Klf4 protein comprises anamino acid sequence having from 1 to 15 or from 1 to 10 amino acidinsertions, deletions, or substitutions (collectively) with respect toSEQ ID NO: 10.

By “c-Myc protein” is meant a protein that is encoded by the MYC gene,or a natural or engineered variant, family-member, orthologue, fragmentor fusion construct thereof, by way of non-limiting example, human c-Mycprotein (SEQ ID NO: 11), mouse c-Myc protein, 1-Myc protein, c-Myc(T58A) protein, a DNA-binding domain of c-Myc protein or a c-Myc-GFPfusion protein. In some embodiments the c-Myc protein comprises an aminoacid sequence that has at least 70% identity with SEQ ID NO: 11, or inother embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQID NO: 11. In some embodiments, the c-Myc protein comprises an aminoacid having from 1 to 20 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 11. Or in otherembodiments, the c-Myc protein comprises an amino acid sequence havingfrom 1 to 15 or from 1 to 10 amino acid insertions, deletions, orsubstitutions (collectively) with respect to SEQ ID NO: 11.

By “reprogramming” is meant causing a change in the phenotype of a cell,by way of non-limiting example, causing a β-cell progenitor todifferentiate into a mature β-cell, causing a fibroblast todedifferentiate into a pluripotent stem cell, causing a keratinocyte totransdifferentiate into a cardiac stem cell or causing the axon of aneuron to grow.

By “reprogramming factor” is meant a molecule that, when a cell iscontacted with the molecule and/or the cell expresses the molecule, can,either alone or in combination with other molecules, causereprogramming, by way of non-limiting example, Oct4 protein.

By “feeder” is meant a cell that can be used to condition medium or tootherwise support the growth of other cells in culture.

By “conditioning” is meant contacting one or more feeders with a medium.

By “fatty acid” is meant a molecule that comprises an aliphatic chain ofat least two carbon atoms, by way of non-limiting example, linoleicacid, α-linolenic acid, octanoic acid, a leukotriene, a prostaglandin,cholesterol, a glucocorticoid, a resolvin, a protectin, a thromboxane, alipoxin, a maresin, a sphingolipid, tryptophan, N-acetyl tryptophan or asalt, methyl ester or derivative thereof.

By “short-chain fatty acid” is meant a fatty acid that comprises analiphatic chain of between two and 30 carbon atoms.

By “albumin” is meant a protein that is highly soluble in water, by wayof non-limiting example, human serum albumin.

By “associated molecule” is meant a molecule that is non-covalentlybound to another molecule.

By “associated-molecule-component of albumin” is meant one or moremolecules that are bound to an albumin polypeptide, by way ofnon-limiting example, lipids, hormones, cholesterol, calcium ions, etc.that are bound to an albumin polypeptide.

By “treated albumin” is meant albumin that is treated to reduce, remove,replace or otherwise inactivate the associated-molecule-component of thealbumin, by way of non-limiting example, human serum albumin that isincubated at an elevated temperature, human serum albumin that iscontacted with sodium octanoate or human serum albumin that is contactedwith a porous material.

By “ion-exchange resin” is meant a material that, when contacted with asolution containing ions, can replace one or more of the ions with oneor more different ions, by way of non-limiting example, a material thatcan replace one or more calcium ions with one or more sodium ions.

By “germ cell” is meant a sperm cell or an egg cell.

By “pluripotent stem cell” is meant a cell that can differentiate intocells of all three germ layers (endoderm, mesoderm, and ectoderm) invivo.

By “somatic cell” is meant a cell that is not a pluripotent stem cell ora germ cell, by way of non-limiting example, a skin cell.

By “glucose-responsive insulin-producing cell” is meant a cell that,when exposed to a certain concentration of glucose, can produce and/orsecrete an amount of insulin that is different from (either less than ormore than) the amount of insulin that the cell produces and/or secreteswhen the cell is exposed to a different concentration of glucose, by wayof non-limiting example, a β-cell.

By “hematopoietic cell” is meant a blood cell or a cell that candifferentiate into a blood cell, by way of non-limiting example, ahematopoietic stem cell or a white blood cell.

By “cardiac cell” is meant a heart cell or a cell that can differentiateinto a heart cell, by way of non-limiting example, a cardiac stem cellor a cardiomyocyte.

By “retinal cell” is meant a cell of the retina or a cell that candifferentiate into a cell of the retina, by way of non-limiting example,a retinal pigmented epithelial cell.

By “skin cell” is meant a cell that is normally found in the skin, byway of non-limiting example, a fibroblast, a keratinocyte, a melanocyte,an adipocyte, a mesenchymal stem cell, an adipose stem cell or a bloodcell.

By “Wnt signaling agonist” is meant a molecule that can perform one ormore of the biological functions of one or more members of the Wntfamily of proteins, by way of non-limiting example, Wnt1, Wnt2, Wnt3,Wnt3a or2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

By “IL-6 signaling agonist” is meant a molecule that can perform one ormore of the biological functions of IL-6 protein, by way of non-limitingexample, IL-6 protein or IL-6 receptor (also known as soluble IL-6receptor, IL-6R, IL-6R alpha, etc.).

By “TGF-β signaling agonist” is meant a molecule that can perform one ormore of the biological functions of one or more members of the TGF-βsuperfamily of proteins, by way of non-limiting example, TGF-β1, TGF-β3,Activin A, BMP-4 or Nodal.

By “immunosuppressant” is meant a substance that can suppress one ormore aspects of an immune system, and that is not normally present in amammal, by way of non-limiting example, B18R or dexamethasone.

By “single-strand break” is meant a region of single-stranded ordouble-stranded DNA in which one or more of the covalent bonds linkingthe nucleotides has been broken in one of the one or two strands.

By “double-strand break” is meant a region of double-stranded DNA inwhich one or more of the covalent bonds linking the nucleotides has beenbroken in each of the two strands.

By “nucleotide” is meant a nucleotide or a fragment or derivativethereof, by way of non-limiting example, a nucleobase, a nucleoside, anucleotide-triphosphate, etc.

By “nucleoside” is meant a nucleotide or a fragment or derivativethereof, by way of non-limiting example, a nucleobase, a nucleoside, anucleotide-triphosphate, etc.

By “gene editing” is meant altering the DNA sequence of a cell, by wayof non-limiting example, by transfecting the cell with a protein thatcauses a mutation in the DNA of the cell.

By “gene-editing protein” is meant a protein that can, either alone orin combination with one or more other molecules, alter the DNA sequenceof a cell, by way of non-limiting example, a nuclease, a transcriptionactivator-like effector nuclease (TALEN), a zinc-finger nuclease, ameganuclease, a nickase, a clustered regularly interspaced shortpalindromic repeat (CRISPR)-associated protein or a natural orengineered variant, family-member, orthologue, fragment or fusionconstruct thereof.

By “repair template” is meant a nucleic acid containing a region of atleast about 70% homology with a sequence that is within 10 kb of atarget site of a gene-editing protein.

By “repeat sequence” is meant an amino-acid sequence that is present inmore than one copy in a protein, to within at least about 10% homology,by way of non-limiting example, a monomer repeat of a transcriptionactivator-like effector.

By “DNA-binding domain” is meant a region of a molecule that is capableof binding to a DNA molecule, by way of non-limiting example, a proteindomain comprising one or more zinc fingers, a protein domain comprisingone or more transcription activator-like (TAL) effector repeat sequencesor a binding pocket of a small molecule that is capable of binding to aDNA molecule.

By “binding site” is meant a nucleic-acid sequence that is capable ofbeing recognized by a gene-editing protein, DNA-binding protein,DNA-binding domain or a biologically active fragment or variant thereofor a nucleic-acid sequence for which a gene-editing protein, DNA-bindingprotein, DNA-binding domain or a biologically active fragment or variantthereof has high affinity, by way of non-limiting example, an about20-base-pair sequence of DNA in exon 1 of the human BIRC5 gene.

By “target” is meant a nucleic acid that contains a binding site.

Other definitions are set forth in U.S. application Ser. No. 13/465,490,U.S. Provisional Application No. 61/664,494, U.S. ProvisionalApplication No. 61/721,302, International Application No.PCT/US12/67966, U.S. Provisional Application No. 61/785,404, and U.S.Provisional Application No. 61/842,874, the contents of which are herebyincorporated by reference in their entireties.

It has now been discovered that the non-canonical nucleotide members ofthe 5-methylcytidine de-methylation pathway, when incorporated intosynthetic RNA, can increase the efficiency with which the synthetic RNAcan be translated into protein, and can decrease the toxicity of thesynthetic RNA.

These non-canonical nucleotides include, for example: 5-methylcytidine,5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine (a.k.a.“cytidine-5-carboxylic acid”). Certain embodiments are thereforedirected to a nucleic acid. In one embodiment, the nucleic acid is asynthetic RNA molecule. In another embodiment, the nucleic acidcomprises one or more non-canonical nucleotides. In one embodiment, thenucleic acid comprises one or more non-canonical nucleotide members ofthe 5-methylcytidine de-methylation pathway. In another embodiment, thenucleic acid comprises at least one of: 5-methylcytidine,5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine or aderivative thereof. In a further embodiment, the nucleic acid comprisesat least one of: pseudouridine, 5-methylpseudouridine, 5-methyluridine,5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine,N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.

5-methylcytidine De-Methylation Pathway

Certain embodiments are directed to a protein. Other embodiments aredirected to a nucleic acid that encodes a protein. In one embodiment,the protein is a protein of interest. In another embodiment, the proteinis selected from: a reprogramming protein and a gene-editing protein. Inone embodiment, the nucleic acid is a plasmid. In another embodiment,the nucleic acid is present in a virus or viral vector. In a furtherembodiment, the virus or viral vector is replication incompetent. In astill further embodiment, the virus or viral vector is replicationcompetent. In one embodiment, the virus or viral vector includes atleast one of: an adenovirus, a retrovirus, a lentivirus, a herpes virus,an adeno-associated virus or a natural or engineered variant thereof,and an engineered virus.

It has also been discovered that certain combinations of non-canonicalnucleotides can be particularly effective at increasing the efficiencywith which synthetic RNA can be translated into protein, and decreasingthe toxicity of synthetic RNA, for example, the combinations:5-methyluridine and 5-methylcytidine, 5-methyluridine and7-deazaguanosine, 5-methylcytidine and 7-deazaguanosine,5-methyluridine, 5-methylcytidine, and 7-deazaguanosine, and5-methyluridine, 5-hydroxymethylcytidine, and 7-deazaguanosine. Certainembodiments are therefore directed to a nucleic acid comprising at leasttwo of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and7-deazaguanosine or one or more derivatives thereof. Other embodimentsare directed to a nucleic acid comprising at least three of:5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and7-deazaguanosine or one or more derivatives thereof. Other embodimentsare directed to a nucleic acid comprising all of: 5-methyluridine,5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or oneor more derivatives thereof. In one embodiment, the nucleic acidcomprises one or more 5-methyluridine residues, one or more5-methylcytidine residues, and one or more 7-deazaguanosine residues orone or more 5-methyluridine residues, one or more5-hydroxymethylcytidine residues, and one or more 7-deazaguanosineresidues.

It has been further discovered that synthetic RNA molecules containingcertain fractions of certain non-canonical nucleotides and combinationsthereof can exhibit particularly high translation efficiency and lowtoxicity. Certain embodiments are therefore directed to a nucleic acidcomprising at least one of: one or more uridine residues, one or morecytidine residues, and one or more guanosine residues, and comprisingone or more non-canonical nucleotides. In one embodiment, between about20% and about 80% of the uridine residues are 5-methyluridine residues.In another embodiment, between about 30% and about 50% of the uridineresidues are 5-methyluridine residues. In a further embodiment, about40% of the uridine residues are 5-methyluridine residues. In oneembodiment, between about 60% and about 80% of the cytidine residues are5-methylcytidine residues. In another embodiment, between about 80% andabout 100% of the cytidine residues are 5-methylcytidine residues. In afurther embodiment, about 100% of the cytidine residues are5-methylcytidine residues. In a still further embodiment, between about20% and about 100% of the cytidine residues are 5-hydroxymethylcytidineresidues. In one embodiment, between about 20% and about 80% of theguanosine residues are 7-deazaguanosine residues. In another embodiment,between about 40% and about 60% of the guanosine residues are7-deazaguanosine residues. In a further embodiment, about 50% of theguanosine residues are 7-deazaguanosine residues. In one embodiment,between about 20% and about 80% or between about 30% and about 60% orabout 40% of the cytidine residues are N4-methylcytidine and/orN4-acetylcytidine residues. In another embodiment, each cytidine residueis a 5-methylcytidine residue. In a further embodiment, about 100% ofthe cytidine residues are 5-methylcytidine residues and/or5-hydroxymethylcytidine residues and/or N4-methylcytidine residuesand/or N4-acetylcytidine residues and/or one or more derivativesthereof. In a still further embodiment, about 40% of the uridineresidues are 5-methyluridine residues, between about 20% and about 100%of the cytidine residues are N4-methylcytidine and/or N4-acetylcytidineresidues, and about 50% of the guanosine residues are 7-deazaguanosineresidues. In one embodiment, about 40% of the uridine residues are5-methyluridine residues and about 100% of the cytidine residues are5-methylcytidine residues. In another embodiment, about 40% of theuridine residues are 5-methyluridine residues and about 50% of theguanosine residues are 7-deazaguanosine residues. In a furtherembodiment, about 100% of the cytidine residues are 5-methylcytidineresidues and about 50% of the guanosine residues are 7-deazaguanosineresidues. In one embodiment, about 40% of the uridine residues are5-methyluridine residues, about 100% of the cytidine residues are5-methylcytidine residues, and about 50% of the guanosine residues are7-deazaguanosine residues. In another embodiment, about 40% of theuridine residues are 5-methyluridine residues, between about 20% andabout 100% of the cytidine residues are 5-hydroxymethylcytidineresidues, and about 50% of the guanosine residues are 7-deazaguanosineresidues. In some embodiments, less than 100% of the cytidine residuesare 5-methylcytidine residues. In other embodiments, less than 100% ofthe cytidine residues are 5-hydroxymethylcytidine residues. In oneembodiment, each uridine residue in the synthetic RNA molecule is apseudouridine residue or a 5-methylpseudouridine residue. In anotherembodiment, about 100% of the uridine residues are pseudouridineresidues and/or 5-methylpseudouridine residues. In a further embodiment,about 100% of the uridine residues are pseudouridine residues and/or5-methylpseudouridine residues, about 100% of the cytidine residues are5-methylcytidine residues, and about 50% of the guanosine residues are7-deazaguanosine residues.

Other non-canonical nucleotides that can be used in place of or incombination with 5-methyluridine include, but are not limited to:pseudouridine and 5-methylpseudouridine (a.k.a. “1-methylpseudouridine”,a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof.Other non-canonical nucleotides that can be used in place of or incombination with 5-methylcytidine and/or 5-hydroxymethylcytidineinclude, but are not limited to: pseudoisocytidine,5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine,5-carboxycytidine, N4-methylcytidine, N4-acetylcytidine or one or morederivatives thereof. In certain embodiments, for example, whenperforming only a single transfection or when the cells beingtransfected are not particularly sensitive to transfection-associatedtoxicity or innate-immune signaling, the fractions of non-canonicalnucleotides can be reduced. Reducing the fraction of non-canonicalnucleotides can be beneficial, in part, because reducing the fraction ofnon-canonical nucleotides can reduce the cost of the nucleic acid. Incertain situations, for example, when minimal immunogenicity of thenucleic acid is desired, the fractions of non-canonical nucleotides canbe increased.

Enzymes such as T7 RNA polymerase may preferentially incorporatecanonical nucleotides in an in vitro-transcription reaction containingboth canonical and non-canonical nucleotides. As a result, an invitro-transcription reaction containing a certain fraction of anon-canonical nucleotide may yield RNA containing a different, oftenlower, fraction of the non-canonical nucleotide than the fraction atwhich the non-canonical nucleotide was present in the reaction. Incertain embodiments, references to nucleotide incorporation fractions(for example, “50% 5-methyluridine”) therefore can refer both to nucleicacids containing the stated fraction of the nucleotide, and to nucleicacids synthesized in a reaction containing the stated fraction of thenucleotide (or nucleotide derivative, for example,nucleotide-triphosphate), even though such a reaction may yield anucleic acid containing a different fraction of the nucleotide than thefraction at which the non-canonical nucleotide was present in thereaction. In addition, different nucleotide sequences can encode thesame protein by utilizing alternative codons. In certain embodiments,references to nucleotide incorporation fractions therefore can referboth to nucleic acids containing the stated fraction of the nucleotide,and to nucleic acids encoding the same protein as a different nucleicacid, wherein the different nucleic acid contains the stated fraction ofthe nucleotide.

The DNA sequence of a cell can be altered by contacting the cell with agene-editing protein or by inducing the cell to express a gene-editingprotein. However, previously disclosed gene-editing proteins suffer fromlow binding efficiency and excessive off-target activity, which canintroduce undesired mutations in the DNA of the cell, severely limitingtheir use in therapeutic applications, in which the introduction ofundesired mutations in a patient's cells could lead to the developmentof cancer. It has now been discovered that gene-editing proteins thatcomprise the StsI endonuclease cleavage domain (SEQ ID NO: 1) canexhibit substantially lower off-target activity than previouslydisclosed gene-editing proteins, while maintaining a high level ofon-target activity. Other novel engineered proteins have also beendiscovered that can exhibit high on-target activity, low off-targetactivity, small size, solubility, and other desirable characteristicswhen they are used as the nuclease domain of a gene-editing protein:StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO:4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQID NO: 7). StsI-HA, StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2(ultra-high activity) can exhibit higher on-target activity than bothwild-type StsI and wild-type FokI, due in part to specific amino-acidsubstitutions within the N-terminal region at the 34 and 61 positions,while StsI-HF (high fidelity) and StsI-UHF (ultra-high fidelity) canexhibit lower off-target activity than both wild-type StsI and wild-typeFokI, due in part to specific amino-acid substitutions within theC-terminal region at the 141 and 152 positions. Certain embodiments aretherefore directed to a protein that comprises a nuclease domain. In oneembodiment, the nuclease domain comprises one or more of: the cleavagedomain of Fold endonuclease (SEQ ID NO: 53), the cleavage domain of StsIendonuclease (SEQ ID NO: 1), StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ IDNO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQID NO: 6), and StsI-UHF (SEQ ID NO: 7) or a biologically active fragmentor variant thereof.

It has also been discovered that engineered gene-editing proteins thatcomprise DNA-binding domains comprising certain novel repeat sequencescan exhibit lower off-target activity than previously disclosedgene-editing proteins, while maintaining a high level of on-targetactivity. Certain of these engineered gene-editing proteins can provideseveral advantages over previously disclosed gene-editing proteins,including, for example, increased flexibility of the linker regionconnecting repeat sequences, which can result in increased bindingefficiency. Certain embodiments are therefore directed to a proteincomprising a plurality of repeat sequences. In one embodiment, at leastone of the repeat sequences contains the amino-acid sequence: GabG,where “a” and “b” each represent any amino acid. In one embodiment, theprotein is a gene-editing protein. In another embodiment, one or more ofthe repeat sequences are present in a DNA-binding domain. In a furtherembodiment, “a” and “b” are each independently selected from the group:H and G. In a still further embodiment, “a” and “b” are H and G,respectively. In one embodiment, the amino-acid sequence is presentwithin about 5 amino acids of the C-terminus of the repeat sequence. Inanother embodiment, the amino-acid sequence is present at the C-terminusof the repeat sequence. In some embodiments, one or more G in theamino-acid sequence GabG is replaced with one or more amino acids otherthan G, for example A, H or GG. In one embodiment, the repeat sequencehas a length of between about 32 and about 40 amino acids or betweenabout 33 and about 39 amino acids or between about 34 and 38 amino acidsor between about 35 and about 37 amino acids or about 36 amino acids orgreater than about 32 amino acids or greater than about 33 amino acidsor greater than about 34 amino acids or greater than about 35 aminoacids. Other embodiments are directed to a protein comprising one ormore transcription activator-like effector domains. In one embodiment,at least one of the transcription activator-like effector domainscomprises a repeat sequence. Other embodiments are directed to a proteincomprising a plurality of repeat sequences generated by inserting one ormore amino acids between at least two of the repeat sequences of atranscription activator-like effector domain. In one embodiment, one ormore amino acids is inserted about 1 or about 2 or about 3 or about 4 orabout 5 amino acids from the C-terminus of at least one repeat sequence.Still other embodiments are directed to a protein comprising a pluralityof repeat sequences, wherein about every other repeat sequence has adifferent length than the repeat sequence immediately preceding orfollowing the repeat sequence. In one embodiment, every other repeatsequence is about 36 amino acids long. In another embodiment, everyother repeat sequence is 36 amino acids long. Still other embodimentsare directed to a protein comprising a plurality of repeat sequences,wherein the plurality of repeat sequences comprises at least two repeatsequences that are each at least 36 amino acids long, and wherein atleast two of the repeat sequences that are at least 36 amino acids longare separated by at least one repeat sequence that is less than 36 aminoacids long. Some embodiments are directed to a protein that comprisesone or more sequences selected from, for example, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, andSEQ ID NO: 60.

Other embodiments are directed to a protein that comprises a DNA-bindingdomain. In some embodiments, the DNA-binding domain comprises aplurality of repeat sequences. In one embodiment, the plurality ofrepeat sequences enables high-specificity recognition of a binding sitein a target DNA molecule. In another embodiment, at least two of therepeat sequences have at least about 50%, or about 60%, or about 70%, orabout 80%, or about 90%, or about 95%, or about 98%, or about 99%homology to each other. In a further embodiment, at least one of therepeat sequences comprises one or more regions capable of binding to abinding site in a target DNA molecule. In a still further embodiment,the binding site comprises a defined sequence of between about 1 toabout 5 bases in length. In one embodiment, the DNA-binding domaincomprises a zinc finger. In another embodiment, the DNA-binding domaincomprises a transcription activator-like effector (TALE). In a furtherembodiment, the plurality of repeat sequences includes at least onerepeat sequence having at least about 50% or about 60% or about 70% orabout 80% or about 90% or about 95% or about 98%, or about 99% homologyto a TALE. In a still further embodiment, the gene-editing proteincomprises a clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein. In one embodiment, the gene-editing proteincomprises a nuclear-localization sequence. In another embodiment, thenuclear-localization sequence comprises the amino-acid sequence:PKKKRKV. In one embodiment, the gene-editing protein comprises amitochondrial-localization sequence. In another embodiment, themitochondrial-localization sequence comprises the amino-acid sequence:LGRVIPRKIASRASLM. In one embodiment, the gene-editing protein comprisesa linker. In another embodiment, the linker connects a DNA-bindingdomain to a nuclease domain. In a further embodiment, the linker isbetween about 1 and about 10 amino acids long. In some embodiments, thelinker is about 1, about 2, or about 3, or about 4, or about 5, or about6, or about 7, or about 8, or about 9, or about 10 amino acids long. Inone embodiment, the gene-editing protein is capable of generating a nickor a double-strand break in a target DNA molecule.

Certain embodiments are directed to a method for modifying the genome ofa cell, the method comprising introducing into the cell a nucleic acidmolecule encoding a non-naturally occurring fusion protein comprising anartificial transcription activator-like (TAL) effector repeat domaincomprising one or more repeat units 36 amino acids in length and anendonuclease domain, wherein the repeat domain is engineered forrecognition of a predetermined nucleotide sequence, and wherein thefusion protein recognizes the predetermined nucleotide sequence. In oneembodiment, the cell is a eukaryotic cell. In another embodiment, thecell is an animal cell. In a further embodiment, the cell is a mammaliancell. In a still further embodiment, the cell is a human cell. In oneembodiment, the cell is a plant cell. In another embodiment, the cell isa prokaryotic cell. In some embodiments, the fusion protein introducesan endonucleolytic cleavage in a nucleic acid of the cell, whereby thegenome of the cell is modified.

Other embodiments are directed to a nucleic acid molecule encoding anon-naturally occurring fusion protein comprising an artificialtranscription activator-like (TAL) effector repeat domain comprising oneor more repeat units 36 amino acids in length and restrictionendonuclease activity, wherein the repeat domain is engineered forrecognition of a predetermined nucleotide sequence and wherein thefusion protein recognizes the predetermined nucleotide sequence. In oneembodiment, the repeat units differ by no more than about seven aminoacids. In another embodiment, each of the repeat units contains theamino acid sequence: LTPXQVVAIAS where X can be either E or Q, and theamino acid sequence: LTPXQVVAIAS is followed on the carboxyl terminus byeither one or two amino acids that determine recognition for one ofadenine, cytosine, guanine or thymine. In one embodiment, the nucleicacid encodes about 1.5 to about 28.5 repeat units. In anotherembodiment, the nucleic acid encodes about 11.5, about 14.5, about 17.5or about 18.5 repeat units. In a further embodiment, the predeterminednucleotide sequence is a promoter region. Some embodiments are directedto a vector comprising a nucleic acid molecule or sequence. In oneembodiment, the vector is a viral vector. In another embodiment, theviral vector comprises one or more of: an adenovirus, a retrovirus, alentivirus, a herpes virus, an adeno-associated virus or a natural orengineered variant thereof, and an engineered virus.

Certain embodiments are directed to a nucleic acid molecule encoding anon-naturally occurring fusion protein comprising a first region thatrecognizes a predetermined nucleotide sequence and a second region withendonuclease activity, wherein the first region contains an artificialTAL effector repeat domain comprising one or more repeat units about 36amino acids in length which differ from each other by no more than sevenamino acids, and wherein the repeat domain is engineered for recognitionof the predetermined nucleotide sequence. In one embodiment, the firstregion contains the amino acid sequence: LTPXQVVAIAS where X can beeither E or Q. In another embodiment, the amino acid sequenceLTPXQVVAIAS of the encoded non-naturally occurring fusion protein isimmediately followed by an amino acid sequence selected from: HD, NG,NS, NI, NN, and N. In a further embodiment, the fusion protein comprisesrestriction endonuclease activity. Some embodiments are directed to anucleic acid molecule encoding a protein that comprises one or moresequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.

In one embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG,wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is any aminoacid or no amino acid, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG orGKQALETVQRLLPVLCQAHG. In another embodiment, the repeat sequencecomprises: LTPvQVVAIAwxyzHG, wherein “v” is D or E, “w” is S or N, “x”is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z”is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG,GKQALETVQRLLPVLCQDHG or GKQALETVQRLLPVLCQAHG. In yet another embodiment,the repeat sequence comprises: LTPvQVVAIAwxyzHG, wherein “v” is D or E,“w” is S or N, “x” is any amino acid other than N, H and I, “y” is anyamino acid or no amino acid, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG orGKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequencecomprises: LTPvQVVAIAwIyzHG, wherein “v” is D or E, “w” is S or N, “y”is any amino acid other than G, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG orGKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequencecomprises: LTPvQVVAIAwIAzHG, wherein “v” is D or E, “w” is S or N, and“z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG,GKQALETVQRLLPVLCQDHG or GKQALETVQRLLPVLCQAHG. In yet another embodiment,the repeat sequence comprises: LTPvQVVAIAwxyzHG, wherein “v” is D or E,“w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid,and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG,GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG or GKQALETVQRLLPVLCQAHG. Inyet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG,wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is selectedfrom: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG orGKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequencecomprises: LTPvQVVAIAwx, wherein “v” is D or E, “w” is S or N, and “x”is S, T or Q. In yet another embodiment, the repeat sequence comprises:LTPvQVVAIAwxy, wherein “v” is D or E, “w” is S or N, “x” is S, T or Q,and “y” is selected from: D, A, I, N, H, K, S, and G. In yet anotherembodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein“v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acidor no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD,GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yetanother embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG,wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” isselected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD orGKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequencecomprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N,“x” is any amino acid other than N, H and I, “y” is any amino acid or noamino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD,GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yetanother embodiment, the repeat sequence comprises: LTPvQVVAIAwIyzGHGG,wherein “v” is Q, D or E, “w” is S or N, “y” is any amino acid otherthan G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD,GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yetanother embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzGHGG,wherein “v” is Q, D or E, “w” is S or N, and “z” is GGRPALE, GGKQALE,GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD orGKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequencecomprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N,“x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” isGGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA,GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, therepeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E,“w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K,S, and G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD,GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yetanother embodiment, the repeat sequence comprises: LTPvQVVAIAwx, wherein“v” is Q, D or E, “w” is S or N, and “x” is S, T or Q. In yet anotherembodiment, the repeat sequence comprises: LTPvQVVAIAwxy, wherein “v” isQ, D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D,A, I, N, H, K, S, and G.

Certain fragments of an endonuclease cleavage domain, includingfragments that are truncated at the N-terminus, fragments that aretruncated at the C-terminus, fragments that have internal deletions, andfragments that combine N-terminus, C-terminus, and/or internaldeletions, can maintain part or all of the catalytic activity of thefull endonuclease cleavage domain. Determining whether a fragment canmaintain part or all of the catalytic activity of the full domain can beaccomplished by, for example, synthesizing a gene-editing protein thatcontains the fragment according to the methods of the present invention,inducing cells to express the gene-editing protein according to themethods of the present invention, and measuring the efficiency of geneediting. In this way, a measurement of gene-editing efficiency can beused to ascertain whether any specific fragment can maintain part or allof the catalytic activity of the full endonuclease cleavage domain.Certain embodiments are therefore directed to a biologically activefragment of an endonuclease cleavage domain. In one embodiment, theendonuclease cleavage domain is selected from: FokI, StsI, StsI-HA,StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural orengineered variant or biologically active fragment thereof.

Certain fragments of a DNA-binding domain or repeat sequence, includingfragments that are truncated at the N-terminus, fragments that aretruncated at the C-terminus, fragments that have internal deletions, andfragments that combine N-terminus, C-terminus, and/or internaldeletions, can maintain part or all of the binding activity of the fullDNA-binding domain or repeat sequence. Examples of fragments ofDNA-binding domains or repeat sequences that can maintain part or all ofthe binding activity of the full repeat sequence include Ralstoniasolanacearum TALE-like proteins (RTLs). Determining whether a fragmentcan maintain part or all of the binding activity of the full DNA-bindingdomain or repeat sequence can be accomplished by, for example,synthesizing a gene-editing protein that contains the fragment accordingto the methods of the present invention, inducing cells to express thegene-editing protein according to the methods of the present invention,and measuring the efficiency of gene editing. In this way, a measurementof gene-editing efficiency can be used to ascertain whether any specificfragment can maintain part or all of the binding activity of the fullDNA-binding domain or repeat sequence. Certain embodiments are thereforedirected to a biologically active fragment of a DNA-binding domain orrepeat sequence. In one embodiment, the fragment enableshigh-specificity recognition of a binding site in a target DNA molecule.In another embodiment, the fragment comprises a sequence that encodes aRalstonia solanacearum TALE-like protein or a biologically activefragment thereof.

Certain embodiments are directed to a composition for altering the DNAsequence of a cell comprising a nucleic acid, wherein the nucleic acidencodes a gene-editing protein. Other embodiments are directed to acomposition for altering the DNA sequence of a cell comprising anucleic-acid mixture, wherein the nucleic-acid mixture comprises: afirst nucleic acid that encodes a first gene-editing protein, and asecond nucleic acid that encodes a second gene-editing protein. In oneembodiment, the binding site of the first gene-editing protein and thebinding site of the second gene-editing protein are present in the sametarget DNA molecule. In another embodiment, the binding site of thefirst gene-editing protein and the binding site of the secondgene-editing protein are separated by less than about 50 bases, or lessthan about 40 bases, or less than about 30 bases or less than about 20bases, or less than about 10 bases, or between about 10 bases and about25 bases or about 15 bases. In one embodiment, the nuclease domain ofthe first gene-editing protein and the nuclease domain of the secondgene-editing protein are capable of forming a dimer. In anotherembodiment, the dimer is capable of generating a nick or double-strandbreak in a target DNA molecule. In one embodiment, the composition is atherapeutic composition. In another embodiment, the compositioncomprises a repair template. In a further embodiment, the repairtemplate is a single-stranded DNA molecule or a double-stranded DNAmolecule.

Other embodiments are directed to an article of manufacture forsynthesizing a protein or a nucleic acid encoding a protein. In oneembodiment, the article is a nucleic acid. In another embodiment, theprotein comprises a DNA-binding domain. In a further embodiment, thenucleic acid comprises a nucleotide sequence encoding a DNA-bindingdomain. In one embodiment, the protein comprises a nuclease domain. Inanother embodiment, the nucleic acid comprises a nucleotide sequenceencoding a nuclease domain. In one embodiment, the protein comprises aplurality of repeat sequences. In another embodiment, the nucleic acidencodes a plurality of repeat sequences. In a further embodiment, thenuclease domain is selected from: FokI, StsI, StsI-HA, StsI-HA2,StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineeredvariant or biologically active fragment thereof. In one embodiment, thenucleic acid comprises an RNA-polymerase promoter. In anotherembodiment, the RNA-polymerase promoter is a T7 promoter or a SP6promoter. In a further embodiment, the nucleic acid comprises a viralpromoter. In one embodiment, the nucleic acid comprises an untranslatedregion. In another embodiment, the nucleic acid is an invitro-transcription template.

Certain embodiments are directed to a method for inducing a cell toexpress a protein. Other embodiments are directed to a method foraltering the DNA sequence of a cell comprising transfecting the cellwith a gene-editing protein or inducing the cell to express agene-editing protein. Still other embodiments are directed to a methodfor reducing the expression of a protein of interest in a cell. In oneembodiment, the cell is induced to express a gene-editing protein,wherein the gene-editing protein is capable of creating a nick or adouble-strand break in a target DNA molecule. In another embodiment, thenick or double-strand break results in inactivation of a gene. Stillother embodiments are directed to a method for generating an inactive,reduced-activity or dominant-negative form of a protein. In oneembodiment, the protein is survivin. Still other embodiments aredirected to a method for repairing one or more mutations in a cell. Inone embodiment, the cell is contacted with a repair template. In anotherembodiment, the repair template is a DNA molecule. In a furtherembodiment, the repair template does not contain a binding site of thegene-editing protein. In a still further embodiment, the repair templateencodes an amino-acid sequence that is encoded by a DNA sequence thatcomprises a binding site of the gene-editing protein.

Other embodiments are directed to a method for treating a patientcomprising administering to the patient a therapeutically effectiveamount of a protein or a nucleic acid encoding a protein. In oneembodiment, the treatment results in one or more of the patient'ssymptoms being ameliorated. Certain embodiments are directed to a methodfor treating a patient comprising: a. removing a cell from the patient,b. inducing the cell to express a gene-editing protein by transfectingthe cell with a nucleic acid encoding a gene-editing protein, c.reprogramming the cell, and e. introducing the cell into the patient. Inone embodiment, the cell is reprogrammed to a less differentiated state.In another embodiment, the cell is reprogrammed by transfecting the cellwith one or more synthetic RNA molecules encoding one or morereprogramming proteins. In a further embodiment, the cell isdifferentiated. In a still further embodiment, the cell isdifferentiated into one of: a skin cell, a glucose-responsiveinsulin-producing cell, a hematopoietic cell, a cardiac cell, a retinalcell, a renal cell, a neural cell, a stromal cell, a fat cell, a bonecell, a muscle cell, an oocyte, and a sperm cell. Other embodiments aredirected to a method for treating a patient comprising: a. removing ahematopoietic cell or a stem cell from the patient, b. inducing the cellto express a gene-editing protein by transfecting the cell with anucleic acid encoding a gene-editing protein, and c. introducing thecell into the patient.

It has now been discovered that a cell-culture medium consistingessentially of or comprising: DMEM/F12, ascorbic acid, insulin,transferrin, sodium selenite, ethanolamine, basic fibroblast growthfactor, and transforming growth factor-beta is sufficient to sustainpluripotent stem cells, including human pluripotent stem cells, invitro. Certain embodiments are therefore directed to a cell-culturemedium consisting essentially of or comprising: DMEM/F12, ascorbic acid,insulin, transferrin, sodium selenite, ethanolamine, basic fibroblastgrowth factor, and transforming growth factor-beta. In one embodiment,the ascorbic acid is present at about 50 μg/mL. In another embodiment,the insulin is present at about 10 μg/mL. In a further embodiment, thetransferrin is present at about 5.5 μg/mL. In a still furtherembodiment, the sodium selenite is present at about 6.7 ng/mL. In astill further embodiment, the ethanolamine is present at about 2 μg/mL.In a still further embodiment, the basic fibroblast growth factor ispresent at about 20 ng/mL. In a still further embodiment, thetransforming growth factor-beta is present at about 2 ng/mL. In oneembodiment, the ascorbic acid is ascorbic acid-2-phosphate. In anotherembodiment, the transforming growth factor-beta is transforming growthfactor-beta 1 or transforming growth factor-beta 3. In one embodiment,the cell-culture medium is used for the culture of pluripotent stemcells. In another embodiment, the pluripotent stem cells are humanpluripotent stem cells. In a further embodiment, the cell-culture mediumis used for the culture of cells during or after reprogramming. In oneembodiment, the cell-culture medium contains no animal-derivedcomponents. In another embodiment, the cell-culture medium ismanufactured according to a manufacturing standard. In a furtherembodiment, the manufacturing standard is GMP. In one embodiment, thecells are contacted with a cell-adhesion molecule. In anotherembodiment, the cell-adhesion molecule is selected from: fibronectin andvitronectin or a biologically active fragment thereof. In a furtherembodiment, the cells are contacted with fibronectin and vitronectin. Ina still further embodiment, the cell-adhesion molecule is recombinant.

In certain situations, for example, when producing a therapeutic, it canbe beneficial to replace animal-derived components withnon-animal-derived components, in part to reduce the risk ofcontamination with viruses and/or other animal-borne pathogens. It hasnow been discovered that synthetic cholesterol, including semi-syntheticplant-derived cholesterol, can be substituted for animal-derivedcholesterol in transfection medium without decreasing transfectionefficiency or increasing transfection-associated toxicity. Certainembodiments are therefore directed to a transfection medium containingsynthetic or semi-synthetic cholesterol. In one embodiment, thesemi-synthetic cholesterol is plant-derived. In another embodiment, thetransfection medium does not contain animal-derived cholesterol. In afurther embodiment, the transfection medium is a reprogramming medium.Other embodiments are directed to a complexation medium. In oneembodiment, the complexation medium has a pH greater than about 7, orgreater than about 7.2, or greater than about 7.4, or greater than about7.6, or greater than about 7.8, or greater than about 8.0, or greaterthan about 8.2, or greater than about 8.4, or greater than about 8.6, orgreater than about 8.8, or greater than about 9.0. In anotherembodiment, the complexation medium comprises transferrin. In a furtherembodiment, the complexation medium comprises DMEM. In a still furtherembodiment, the complexation medium comprises DMEM/F12. Still otherembodiments are directed to a method for formingnucleic-acid-transfection-reagent complexes. In one embodiment, thetransfection reagent is incubated with a complexation medium. In anotherembodiment, the incubation occurs before a mixing step. In a furtherembodiment, the incubation step is between about 5 seconds and about 5minutes or between about 10 seconds and about 2 minutes or between about15 seconds and about 1 minute or between about 30 seconds and about 45seconds. In one embodiment, the transfection reagent is selected fromTable 1. In another embodiment, the transfection reagent is a lipid orlipidoid. In a further embodiment, the transfection reagent comprises acation. In a still further embodiment, the cation is a multivalentcation. In a still further embodiment, the transfection reagent isN1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(a.k.a. MVL5) or a derivative thereof.

Certain embodiments are directed to a method for inducing a cell toexpress a protein by contacting the cell with a nucleic acid. In oneembodiment, the cell is a mammalian cell. In another embodiment, thecell is a human cell or a rodent cell. Other embodiments are directed toa cell produced using one or more of the methods of the presentinvention. In one embodiment, the cell is present in a patient. Inanother embodiment, the cell is isolated from a patient. Otherembodiments are directed to a screening library comprising a cellproduced using one or more of the methods of the present invention. Inone embodiment, the screening library is used for at least one of:toxicity screening, including: cardiotoxicity screening, neurotoxicityscreening, and hepatotoxicity screening, efficacy screening,high-throughput screening, high-content screening, and other screening.

Other embodiments are directed to a kit containing a nucleic acid. Inone embodiment, the kit contains a delivery reagent (a.k.a.“transfection reagent”). In another embodiment, the kit is areprogramming kit. In a further embodiment, the kit is a gene-editingkit. Other embodiments are directed to a kit for producing nucleicacids. In one embodiment, the kit contains at least two of:pseudouridine-triphosphate, 5-methyluridine triphosphate,5-methylcytidine triphosphate, 5-hydroxymethylcytidine triphosphate,N4-methylcytidine triphosphate, N4-acetylcytidine triphosphate, and7-deazaguanosine triphosphate or one or more derivatives thereof. Otherembodiments are directed to a therapeutic comprising a nucleic acid. Inone embodiment, the therapeutic is a pharmaceutical composition. Inanother embodiment, the pharmaceutical composition is formulated. In afurther embodiment, the formulation comprises an aqueous suspension ofliposomes. Example liposome components are set forth in Table 1, and aregiven by way of example, and not by way of limitation. In oneembodiment, the liposomes include one or more polyethylene glycol (PEG)chains. In another embodiment, the PEG is PEG2000. In a furtherembodiment, the liposomes include1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivativethereof. In one embodiment, the therapeutic comprises one or moreligands. In another embodiment, the therapeutic comprises at least oneof: androgen, CD30 (TNFRSF8), a cell-penetrating peptide, CXCR,estrogen, epidermal growth factor, EGFR, HER2, folate, insulin,insulin-like growth factor-I, interleukin-13, integrin, progesterone,stromal-derived-factor-1, thrombin, vitamin D, and transferrin or abiologically active fragment or variant thereof. Still other embodimentsare directed to a therapeutic comprising a cell generated using one ormore of the methods of the present invention. In one embodiment, thetherapeutic is administered to a patient for the treatment of at leastone of: type 1 diabetes, heart disease, including ischemic and dilatedcardiomyopathy, macular degeneration, Parkinson's disease, cysticfibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severecombined immunodeficiency, hereditary sensory neuropathy, xerodermapigmentosum, Huntington's disease, muscular dystrophy, amyotrophiclateral sclerosis, Alzheimer's disease, cancer, and infectious diseasesincluding: hepatitis and HIV/AIDS.

TABLE 1 Exemplary Biocompatible Lipids 13β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol)2 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP/18:1 TAP) 3N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(DOBAQ) 4 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) 51,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP) 61,2-stearoyl-3-trimethylammonium-propane (18:0 TAP) 71,2-dioleoyl-3-dimethylammonium-propane (DODAP/18:1 DAP) 81,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP) 91,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP) 101,2-distearoyl-3-dimethylammonium-propane (18:0 DAP) 11dimethyldioctadecylammonium (18:0 DDAB) 121,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EthylPC) 131,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EthylPC) 141,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EthylPC) 151,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EthylPC) 161,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EthylPC) 171,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EthylPC) 18 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:1-18:1 EthylPC)19 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) 20N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5) 21 2,3-dioleyloxy-N-[2-sperminecarboxamide]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate (DOSPA)22 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER) 23N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammoniumbromide (DMRIE) 24 dioctadecyl amidoglyceryl spermine (DOGS) 25 dioleoylphosphatidyl ethanolamine (DOPE)

Certain embodiments are directed to a nucleic acid comprising a 5′-capstructure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivativethereof. In one embodiment, the nucleic acid comprises one or more UTRs.In another embodiment, the one or more UTRs increase the stability ofthe nucleic acid. In a further embodiment, the one or more UTRs comprisean alpha-globin or beta-globin 5′-UTR. In a still further embodiment,the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. Ina still further embodiment, the synthetic RNA molecule comprises analpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that issubstantially similar to the Kozak consensus sequence. In anotherembodiment, the nucleic acid comprises a 3′-poly(A) tail. In a furtherembodiment, the 3′-poly(A) tail is between about 20 nt and about 250 ntor between about 120 nt and about 150 nt long. In a further embodiment,the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, orabout 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt,or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, orabout 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, orabout 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

Other embodiments are directed to a method for reprogramming a cell. Inone embodiment, the cell is reprogrammed by contacting the cell with oneor more nucleic acids. In one embodiment, the cell is contacted with aplurality of nucleic acids encoding at least one of: Oct4 protein, Sox2protein, Klf4 protein, c-Myc protein, Lin28 protein or a biologicallyactive fragment, variant or derivative thereof. In another embodiment,the cell is contacted with a plurality of nucleic acids encoding aplurality of proteins including: Oct4 protein, Sox2 protein, Klf4protein, and c-Myc protein or one or more biologically active fragments,variants or derivatives thereof. Still other embodiments are directed toa method for gene editing a cell. In one embodiment, the cell isgene-edited by contacting the cell with one or more nucleic acids.

Animal models are routinely used to study the effects of biologicalprocesses. In certain situations, for example, when studying a humandisease, an animal model containing a modified genome can be beneficial,in part because such an animal model may more closely mimic the humandisease phenotype. Certain embodiments are therefore directed to amethod for creating an organism containing one or more geneticmodifications (a.k.a. “mutations”, a.k.a. “gene edits”). In oneembodiment, the one or more genetic modifications is generated bytransfecting a cell with one or more nucleic acids encoding one or moregene-editing proteins. In another embodiment, the one or more nucleicacids include a synthetic RNA molecule. In one embodiment, the one ormore gene-editing proteins include at least one of: a zinc fingernuclease, a TALEN, a clustered regularly interspaced short palindromicrepeat (CRISPR)-associated protein, a nuclease, a meganuclease, and anickase or a biologically active fragment or variant thereof. In oneembodiment, the cell is a pluripotent cell. In another embodiment, thecell is an embryonic stem cell. In a further embodiment, the cell is anembryo. In a still further embodiment, the cell is a member of: ananimal cell, a plant cell, a yeast cell, and a bacterial cell. In oneembodiment, the cell is a rodent cell. In another embodiment, the cellis a human cell. In certain embodiments, the cell is transfected withone or more nucleic acids encoding one or more gene-editing proteins andone or more nucleic acids encoding one or more repair templates. In oneembodiment, the cell is introduced into a blastocyst. In anotherembodiment, the cell is introduced into a pseudopregnant female. In afurther embodiment, the presence or absence of the genetic modificationin the offspring is determined. In a still further embodiment, thedetermining is by direct sequencing. In one embodiment, the organism islivestock, for example, a pig, a cow, etc. In another embodiment, theorganism is a pet, for example, a dog, a cat, a fish, etc.

In certain situations, for example, when modifying the genome of atarget cell by the addition of a nucleic-acid sequence, it can beadvantageous to insert the nucleic-acid sequence into a safe-harborlocation, in part to reduce the risks associated with random insertion.Certain embodiments are therefore directed to a method for inserting anucleic-acid sequence into a safe-harbor location. In one embodiment,the cell is a human cell and the safe-harbor location is the AAVS1locus. In another embodiment, the cell is a rodent cell and thesafe-harbor location is the Rosa26 locus. In one embodiment, the cell isfurther contacted with one or more nucleic acids encoding one or morerepair templates. Other embodiments are directed to a kit for alteringthe DNA sequence of a cell. In one embodiment, the cell is a human cell,and the target DNA molecule comprises a nucleotide sequence that encodesthe AAVS1 locus. In another embodiment, the cell is a rodent cell, andthe target DNA molecule comprises a nucleotide sequence that encodes theRosa26 locus. Other embodiments are directed to a method for generatinga reporter cell by contacting the cell with one or more nucleic acidsencoding one or more gene-editing proteins and one or more nucleic acidsencoding one or more repair templates. In one embodiment, the one ormore repair templates comprise DNA. In another embodiment, the one ormore repair templates encode one or more fluorescent proteins. In afurther embodiment, the one or more repair templates encode at leastpart of the promoter region of a gene.

In certain situations, for example, when generating a library ofgene-edited cells, it can be beneficial to increase the efficiency ofgene editing, in part to reduce the cost of cell characterization. Ithas now been discovered that gene-editing efficiency can be increased byrepeatedly contacting a cell with synthetic RNA encoding one or moregene-editing proteins. Certain embodiments are therefore directed to amethod for gene editing a cell by repeatedly contacting the cell withone or more nucleic acids encoding one or more gene-editing proteins. Inone embodiment, the cell is contacted at least twice during fiveconsecutive days. In another embodiment, the cell is contacted twice atan interval of between about 24 hours and about 48 hours.

In cancer, the survival and proliferation of malignant cells can be duein part to the presence of specific genetic abnormalities that are notgenerally present in the patient. It has now been discovered thatgene-editing proteins can be used to target survival andproliferation-associated pathways, and that when used in this manner,gene-editing proteins and nucleic acids encoding gene-editing proteinscan constitute potent anti-cancer therapeutics. Certain embodiments aretherefore directed to an anti-cancer therapeutic. In one embodiment, thetherapeutic is a therapeutic composition that inhibits the survivaland/or prevents, slows or otherwise limits the proliferation of a cell.In another embodiment, the cell is a cancer cell. In a furtherembodiment, the therapeutic comprises one or more gene-editing proteinsor a nucleic acid that encodes one or more gene-editing proteins. In astill further embodiment, the one or more gene-editing proteins targetone or more sequences that promote survival and/or proliferation of thecell. Such sequences include, but are not limited to: apoptosis-relatedgenes, including genes of the inhibitor of apoptosis (IAP) family (See,e.g., Table 2 and Table 2 of U.S. Provisional Application No.61/721,302, the contents of which are hereby incorporated by reference),such as BIRC5, sequences associated with telomere maintenance, such asthe gene telomerase reverse transcriptase (TERT) and the telomerase RNAcomponent (TERC), sequences affecting angiogenesis, such as the geneVEGF, and other cancer-associated genes, including: BRAF, BRCA1, BRCA2,CDKN2A, CTNNB1, EGFR, the MYC family, the RAS family, PIK3CA, PIK3R1,PKN3, TP53, PTEN, RET, SMAD4, KIT, MET, APC, RB1, the VEGF family, TNF,and genes of the ribonucleotide reductase family. Example gene-editingprotein target sequences for BIRC5 are set forth in Table 3 and in Table3 of U.S. Provisional Application No. 61/721,302, the contents of whichare hereby incorporated by reference, and are given by way of example,and not by way of limitation. In one embodiment, at least one of the oneor more sequences is present in both malignant and non-malignant cells.In another embodiment, at least one of the one or more sequences isenriched in malignant cells. In a further embodiment, at least one ofthe one or more sequences is enriched in non-malignant cells. In oneembodiment, the therapeutic composition further comprises a nucleic acidencoding one or more repair templates. In another embodiment, the one ormore gene-editing proteins induce the cells to express an inactive ordominant-negative form of a protein. In a further embodiment, theprotein is a member of the IAP family. In a still further embodiment,the protein is survivin.

TABLE 2 Exemplary Inhibitor of Apoptosis (IAP) Genes BIR CARD RING NameLength/aa Domains Domain Domain BIRC1 (neuronal 1,403 3 N Napoptosis-inhibitory protein) BIRC2 (c-IAP1 protein) 604 3 Y Y BIRC3(c-IAP2 protein) 618 3 Y Y BIRC4 (X-linked IAP) 497 3 N Y BIRC5(survivin protein) 142 1 N N BIRC6 (BRUCE/apollon 4845 1 N N protein)BIRC7 (livin protein) 298 1 N Y ILP2 (tissue-specific 236 1 N Y homologof BIRC4)

TABLE 3 Exemplary Gene Editing-Protein Target Sequences for BIRC5 TargetLeft Right UTR TAAGAGGGCGTGCGCTCCCG TCAAATCTGGCGGTTAATGG StartTTGGCAGAGGTGGCGGCGGC TGCCAGGCAGGGGGCAACGT Codon Exon 1TTGCCCCCTGCCTGGCAGCC TTCTTGAATGTAGAGATGCG Exon 2 TCCACTGCCCCACTGAGAACTCCTTGAAGCAGAAGAAACA Exon 4 TAAAAAGCATTCGTCCGGTT TTCTTCAAACTGCTTCTTGAExon 5 TTGAGGAAACTGCGGAGAAA TCCATGGCAGCCAGCTGCTC

Other embodiments are directed to a method for treating cancercomprising administering to a patient a therapeutically effective amountof a gene-editing protein or a nucleic acid encoding one or moregene-editing proteins. In one embodiment, the treatment results in thegrowth of cancer cells in the patient being reduced or halted. Inanother embodiment, the treatment results in delayed progression orremission of the cancer. In one embodiment, the target DNA moleculecomprises the BIRC5 gene. In another embodiment, the target DNA moleculecomprises a sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, and SEQ ID NO: 15. In a further embodiment, a plurality ofadjacent binding sites are at least about 50% or at least about 60% orat least about 70% or at least about 80% or at least about 90% or atleast about 95% or at least about 98%, or at least about 99% homologousto one or more sequences listed in Table 3, Table 4, Table 3 of U.S.Provisional Application No. 61/721,302, the contents of which are herebyincorporated by reference, Table 1 of U.S. Provisional Application No.61/785,404, the contents of which are hereby incorporated by referenceor Table 1 of U.S. Provisional Application No. 61/842,874, the contentsof which are hereby incorporated by reference. In certain situations, agene-editing protein with a truncated N-terminal domain can be used toeliminate the first-base-T restriction on the binding-site sequence. Insome embodiments, the cancer is glioma. In one embodiment, the patienthas previously undergone surgery and/or radiation therapy and/orconcurrently undergoes surgery and/or radiation therapy. In anotherembodiment, the administering is by one or more of: intrathecalinjection, intracranial injection, intravenous injection, perfusion,subcutaneous injection, intraperitoneal injection, intraportalinjection, and topical delivery.

TABLE 4 Exemplary BIRC5 Binding Sites Gene # Left Right Spacing BIRC5 1TGGGTGCCCCGACGTTGCCC TGCGGTGGTCCTTGAGAAAG 14 BIRC5 2TGGGTGCCCCGACGTTGCCC TAGAGATGCGGTGGTCCTTG 20 BIRC5 3TGCCCCGACGTTGCCCCCTG TAGAGATGCGGTGGTCCTTG 16 BIRC5 4TGCCCCGACGTTGCCCCCTG TGTAGAGATGCGGTGGTCCT 18 BIRC5 5TCAAGGACCACCGCATCTCT TGCAGGCGCAGCCCTCCAAG 20 BIRC5 6TCTCTACATTCAAGAACTGG TCACCCGCTCCGGGGTGCAG 20 BIRC5 7TCTACATTCAAGAACTGGCC TCACCCGCTCCGGGGTGCAG 18 BIRC5 8TCTACATTCAAGAACTGGCC TCTCACCCGCTCCGGGGTGC 20 BIRC5 9TACATTCAAGAACTGGCCCT TCACCCGCTCCGGGGTGCAG 16 BIRC5 10TACATTCAAGAACTGGCCCT TCTCACCCGCTCCGGGGTGC 18 BIRC5 11TTCAAGAACTGGCCCTTCTT TCTCACCCGCTCCGGGGTGC 14 BIRC5 1TCCCTTGCAGATGGCCGAGG TGGCTCGTTCTCAGTGGGGC 15 BIRC5 2TCCCTTGCAGATGGCCGAGG TCTGGCTCGTTCTCAGTGGG 17 BIRC5 3TGGCCGAGGCTGGCTTCATC TGGGCCAAGTCTGGCTCGTT 15 BIRC5 4TCCACTGCCCCACTGAGAAC TCCTTGAAGCAGAAGAAACA 18 BIRC5 5TGCCCCACTGAGAACGAGCC TCCAGCTCCTTGAAGCAGAA 19 BIRC5 6TGCCCCACTGAGAACGAGCC TTCCAGCTCCTTGAAGCAGA 20 BIRC5 7TTGGCCCAGTGTTTCTTCTG TCGTCATCTGGCTCCCAGCC 16 BIRC5 8TGGCCCAGTGTTTCTTCTGC TCGTCATCTGGCTCCCAGCC 15 BIRC5 9TGGCCCAGTGTTTCTTCTGC TGGGGTCGTCATCTGGCTCC 20 BIRC5 10TGTTTCTTCTGCTTCAAGGA TACATGGGGTCGTCATCTGG 16 BIRC5 11TGTTTCTTCTGCTTCAAGGA TTACATGGGGTCGTCATCTG 17 BIRC5 12TTTCTTCTGCTTCAAGGAGC TACATGGGGTCGTCATCTGG 14 BIRC5 13TTTCTTCTGCTTCAAGGAGC TTACATGGGGTCGTCATCTG 15 BIRC5 14TTCTTCTGCTTCAAGGAGCT TTACATGGGGTCGTCATCTG 14 BIRC5 1TTTTCTAGAGAGGAACATAA TGACAGAAAGGAAAGCGCAA 15 BIRC5 2TTTTCTAGAGAGGAACATAA TTGACAGAAAGGAAAGCGCA 16 BIRC5 3TTTTCTAGAGAGGAACATAA TCTTGACAGAAAGGAAAGCG 18 BIRC5 4TAGAGAGGAACATAAAAAGC TGCTTCTTGACAGAAAGGAA 17 BIRC5 5TAAAAAGCATTCGTCCGGTT TCTTCAAACTGCTTCTTGAC 14 BIRC5 6TAAAAAGCATTCGTCCGGTT TTCTTCAAACTGCTTCTTGA 15 BIRC5 7TAAAAAGCATTCGTCCGGTT TAATTCTTCAAACTGCTTCT 18 BIRC5 8TAAAAAGCATTCGTCCGGTT TTAATTCTTCAAACTGCTTC 19 BIRC5 9TTCGTCCGGTTGCGCTTTCC TCACCAAGGGTTAATTCTTC 20 BIRC5 10TCGTCCGGTTGCGCTTTCCT TCACCAAGGGTTAATTCTTC 19 BIRC5 11TCGTCCGGTTGCGCTTTCCT TTCACCAAGGGTTAATTCTT 20 BIRC5 12TCCGGTTGCGCTTTCCTTTC TCACCAAGGGTTAATTCTTC 16 BIRC5 13TCCGGTTGCGCTTTCCTTTC TTCACCAAGGGTTAATTCTT 17 BIRC5 14TTGCGCTTTCCTTTCTGTCA TCAAAAATTCACCAAGGGTT 19 BIRC5 15TTGCGCTTTCCTTTCTGTCA TTCAAAAATTCACCAAGGGT 20 BIRC5 16TGCGCTTTCCTTTCTGTCAA TCAAAAATTCACCAAGGGTT 18 BIRC5 17TGCGCTTTCCTTTCTGTCAA TTCAAAAATTCACCAAGGGT 19 BIRC5 18TGCGCTTTCCTTTCTGTCAA TTTCAAAAATTCACCAAGGG 20 BIRC5 19TTTCCTTTCTGTCAAGAAGC TTCAAAAATTCACCAAGGGT 14 BIRC5 20TTTCCTTTCTGTCAAGAAGC TTTCAAAAATTCACCAAGGG 15 BIRC5 21TTTCCTTTCTGTCAAGAAGC TCCAGTTTCAAAAATTCACC 20 BIRC5 22TTCCTTTCTGTCAAGAAGCA TTTCAAAAATTCACCAAGGG 14 BIRC5 23TTCCTTTCTGTCAAGAAGCA TCCAGTTTCAAAAATTCACC 19 BIRC5 24TCCTTTCTGTCAAGAAGCAG TCCAGTTTCAAAAATTCACC 18 BIRC5 25TCCTTTCTGTCAAGAAGCAG TGTCCAGTTTCAAAAATTCA 20 BIRC5 26TTTCTGTCAAGAAGCAGTTT TCCAGTTTCAAAAATTCACC 15 BIRC5 27TTTCTGTCAAGAAGCAGTTT TGTCCAGTTTCAAAAATTCA 17 BIRC5 28TTTCTGTCAAGAAGCAGTTT TCTGTCCAGTTTCAAAAATT 19 BIRC5 29TTCTGTCAAGAAGCAGTTTG TCCAGTTTCAAAAATTCACC 14 BIRC5 30TTCTGTCAAGAAGCAGTTTG TGTCCAGTTTCAAAAATTCA 16 BIRC5 31TTCTGTCAAGAAGCAGTTTG TCTGTCCAGTTTCAAAAATT 18 BIRC5 32TTCTGTCAAGAAGCAGTTTG TCTCTGTCCAGTTTCAAAAA 20 BIRC5 33TCTGTCAAGAAGCAGTTTGA TGTCCAGTTTCAAAAATTCA 15 BIRC5 34TCTGTCAAGAAGCAGTTTGA TCTGTCCAGTTTCAAAAATT 17 BIRC5 35TCTGTCAAGAAGCAGTTTGA TCTCTGTCCAGTTTCAAAAA 19 BIRC5 36TCTGTCAAGAAGCAGTTTGA TTCTCTGTCCAGTTTCAAAA 20 BIRC5 37TGTCAAGAAGCAGTTTGAAG TCTGTCCAGTTTCAAAAATT 15 BIRC5 38TGTCAAGAAGCAGTTTGAAG TCTCTGTCCAGTTTCAAAAA 17 BIRC5 39TGTCAAGAAGCAGTTTGAAG TTCTCTGTCCAGTTTCAAAA 18 BIRC5 40TGTCAAGAAGCAGTTTGAAG TTTCTCTGTCCAGTTTCAAA 19 BIRC5 41TCAAGAAGCAGTTTGAAGAA TCTCTGTCCAGTTTCAAAAA 15 BIRC5 42TCAAGAAGCAGTTTGAAGAA TTCTCTGTCCAGTTTCAAAA 16 BIRC5 43TCAAGAAGCAGTTTGAAGAA TTTCTCTGTCCAGTTTCAAA 17 BIRC5 44TTTGAAGAATTAACCCTTGG TCTTGGCTCTTTCTCTGTCC 15 BIRC5 45TTGAAGAATTAACCCTTGGT TCTTGGCTCTTTCTCTGTCC 14 BIRC5 46TTGAAGAATTAACCCTTGGT TTCTTGGCTCTTTCTCTGTC 15 BIRC5 47TGAAGAATTAACCCTTGGTG TTCTTGGCTCTTTCTCTGTC 14 BIRC5 48TGAAGAATTAACCCTTGGTG TGTTCTTGGCTCTTTCTCTG 16 BIRC5 49TTAACCCTTGGTGAATTTTT TACAATTTTGTTCTTGGCTC 17 BIRC5 50TAACCCTTGGTGAATTTTTG TACAATTTTGTTCTTGGCTC 16 BIRC5 51TAACCCTTGGTGAATTTTTG TACATACAATTTTGTTCTTG 20 BIRC5 52TTGGTGAATTTTTGAAACTG TACATACAATTTTGTTCTTG 14 BIRC5 1TTATTTCCAGGCAAAGGAAA TCCGCAGTTTCCTCAAATTC 17 BIRC5 2TTATTTCCAGGCAAAGGAAA TCTCCGCAGTTTCCTCAAAT 19 BIRC5 3TTATTTCCAGGCAAAGGAAA TTCTCCGCAGTTTCCTCAAA 20 BIRC5 4TATTTCCAGGCAAAGGAAAC TCCGCAGTTTCCTCAAATTC 16 BIRC5 5TATTTCCAGGCAAAGGAAAC TCTCCGCAGTTTCCTCAAAT 18 BIRC5 6TATTTCCAGGCAAAGGAAAC TTCTCCGCAGTTTCCTCAAA 19 BIRC5 7TATTTCCAGGCAAAGGAAAC TTTCTCCGCAGTTTCCTCAA 20 BIRC5 8TCCAGGCAAAGGAAACCAAC TCTCCGCAGTTTCCTCAAAT 14 BIRC5 9TCCAGGCAAAGGAAACCAAC TTCTCCGCAGTTTCCTCAAA 15 BIRC5 10TCCAGGCAAAGGAAACCAAC TTTCTCCGCAGTTTCCTCAA 16 BIRC5 11TTTGAGGAAACTGCGGAGAA TCCATGGCAGCCAGCTGCTC 16 BIRC5 12TTTGAGGAAACTGCGGAGAA TCAATCCATGGCAGCCAGCT 20 BIRC5 13TTGAGGAAACTGCGGAGAAA TCCATGGCAGCCAGCTGCTC 15 BIRC5 14TTGAGGAAACTGCGGAGAAA TCAATCCATGGCAGCCAGCT 19 BIRC5 15TGAGGAAACTGCGGAGAAAG TCCATGGCAGCCAGCTGCTC 14 BIRC5 16TGAGGAAACTGCGGAGAAAG TCAATCCATGGCAGCCAGCT 18

Certain embodiments are directed to a method for treating cancercomprising: a. removing a biopsy containing one or more cancerous cellsfrom a patient, b. determining the sequence of a cancer-associatedgenetic marker in the one or more cancerous cells, and c. administeringto the patient a therapeutically effective amount of a gene-editingprotein or a nucleic acid encoding a gene-editing protein, wherein thesequence of the target DNA molecule is at least about 50% or about 60%or about 70% or about 80% or about 90% or about 95% or about 98%, orabout 99% homologous to the sequence of the cancer-associated geneticmarker. In one embodiment, the method further comprises comparing thesequence of one or more cancer-associated genetic markers in the one ormore cancerous cells to the sequence of the same cancer-associatedgenetic markers in one or more non-cancerous cells, selecting acancer-associated genetic marker having a sequence that is different inthe one or more cancerous cells and the one or more non-cancerous cells,and wherein the sequence of the target DNA molecule or binding site isat least about 50% or about 60% or about 70% or about 80% or about 90%or about 95% or about 98% or about 99% homologous to the sequence of theselected cancer-associated genetic marker.

Many cancer cells express survivin, a member of the inhibitor ofapoptosis (IAP) protein family that, in humans, is encoded by the BIRC5gene. Using RNA interference to reduce expression of certain mRNAmolecules, including survivin mRNA, can transiently inhibit the growthof certain cancer cells. However, previous methods of using RNAinterference to reduce expression of survivin mRNA yield temporaryeffects, and result in only a short increase in mean time-to-death (TTD)in animal models. It has now been discovered that inducing a cell toexpress one or more gene-editing proteins that target the BIRC5 gene canresult in disruption of the BIRC5 gene, can induce the cell to expressand/or secrete a non-functional variant of survivin protein, can inducethe cell to express and/or secrete a dominant-negative variant ofsurvivin protein, can trigger activation of one or more apoptosispathways in the cell and nearby cells, can slow or halt the growth ofthe cell and nearby cells, can result in the death of the cell andnearby cells, can inhibit the progression of cancer, and can result inremission in a cancer patient. Certain embodiments are thereforedirected to a gene-editing protein that targets the BIRC5 gene. In oneembodiment, the gene-editing protein binds to one or more regions in theBIRC5 gene. In another embodiment, the gene-editing protein binds to oneor more regions of a sequence selected from: SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, and SEQ ID NO: 15. In a further embodiment, thegene-editing protein binds to one or more sequences selected from: SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, and SEQ ID NO: 27. In a still further embodiment, thegene-editing protein binds to one or more nucleic-acid sequences thatencode SEQ ID NO: 34 or a biologically active fragment, variant oranalogue thereof. In a still further embodiment, the gene-editingprotein binds to one or more sequences selected from Table 3, Table 4,Table 3 of U.S. Provisional Application No. 61/721,302, the contents ofwhich are hereby incorporated by reference, Table 1 of U.S. ProvisionalApplication No. 61/785,404, the contents of which are herebyincorporated by reference or Table 1 of U.S. Provisional Application No.61/842,874, the contents of which are hereby incorporated by referenceor to one or more sequences that is at least about 50% or at least about60% or at least about 70% or at least about 80% or at least about 90% orat least about 95% or at least about 98%, or about 99% homologous to oneor more sequences selected from Table 3, Table 4, Table 3 of U.S.Provisional Application No. 61/721,302, the contents of which are herebyincorporated by reference, Table 1 of U.S. Provisional Application No.61/785,404, the contents of which are hereby incorporated by referenceor Table 1 of U.S. Provisional Application No. 61/842,874, the contentsof which are hereby incorporated by reference. In one embodiment, thegene-editing protein creates one or more nicks or double-strand breaksin the DNA of the cell. In another embodiment, the one or more nicks ordouble-strand breaks is created in the BIRC5 gene. In a furtherembodiment, the one or more nicks or double-strand breaks is created inone or more exons of the BIRC5 gene. In a still further embodiment, theone or more nicks or double-strand breaks is created in a sequenceselected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ IDNO: 15. In a still further embodiment, the one or more nicks ordouble-strand breaks is created within a sequence that encodes aninhibitor of apoptosis domain (aka. “IAP”, “IAP domain”, “IAP repeat”,“baculovirus inhibitor of apoptosis protein repeat”, “BIR”, etc.). In astill further embodiment, the gene-editing protein binds to one or moresequences selected from Table 5, Table 2 of U.S. Provisional ApplicationNo. 61/785,404, the contents of which are hereby incorporated byreference or Table 2 of U.S. Provisional Application No. 61/842,874, thecontents of which are hereby incorporated by reference or to one or moresequences that is at least about 50% or at least about 60% or at leastabout 70% or at least about 80% or at least about 90% or at least about95% or at least about 98% homologous to one or more sequences selectedfrom Table 5, Table 2 of U.S. Provisional Application No. 61/785,404,the contents of which are hereby incorporated by reference or Table 2 ofU.S. Provisional Application No. 61/842,874, the contents of which arehereby incorporated by reference. In yet another embodiment, the geneediting protein binds to a sequence that encodes one or more genesselected from Table 2, Table 5, Table 6, Table 7, Table 4 of U.S.Provisional Application No. 61/721,302, the contents of which are herebyincorporated by reference, Table 2 of U.S. Provisional Application No.61/785,404, the contents of which are hereby incorporated by referenceor Table 2 of U.S. Provisional Application No. 61/842,874, the contentsof which are hereby incorporated by reference.

TABLE 5 Exemplary Cancer-Associated Gene Binding Sites Gene # Left RightSpacing CDK1 1 TTTAGGATCTACCATACCCA TCTCTATTTTGGTATAATCT 15 CDK1 2TTTAGGATCTACCATACCCA TTCTCTATTTTGGTATAATC 16 CDK1 3 TTTAGGATCTACCATACCCATTTCTCTATTTTGGTATAAT 17 CDK1 4 TTAGGATCTACCATACCCAT TCTCTATTTTGGTATAATCT14 CDK1 5 TTAGGATCTACCATACCCAT TTCTCTATTTTGGTATAATC 15 CDK1 1TCACACAGCATATTATTTAC TACCCTTATACACAACTCCA 17 CDK1 2 TCACACAGCATATTATTTACTCTACCCTTATACACAACTC 19 CDK1 3 TACTTTGTTTCAGGTACCTA TGTAGTTTTGTGTCTACCCT14 CDK1 4 TACTTTGTTTCAGGTACCTA TGACCTGTAGTTTTGTGTCT 19 CDK1 5TTTGTTTCAGGTACCTATGG TGACCTGTAGTTTTGTGTCT 16 CDK2 1 TGACCCGACTCGCTGGCGCTTCCGATCTTTTCCACCTTTT 15 CDK2 2 TGACCCGACTCGCTGGCGCT TCTCCGATCTTTTCCACCTT17 CDK2 3 TCGCTGGCGCTTCATGGAGA TACGTGCCCTCTCCGATCTT 17 CDK2 4TTCATGGAGAACTTCCAAAA TACACAACTCCGTACGTGCC 19 CDK2 5 TCATGGAGAACTTCCAAAAGTACACAACTCCGTACGTGCC 18 CDK2 1 TTTCCCAACCTCTCCAAGTG TCTCGGATGGCAGTACTGGG14 CDK2 2 TTCCCAACCTCTCCAAGTGA TCTCTCGGATGGCAGTACTG 15 CDK2 3TCCCAACCTCTCCAAGTGAG TCTCTCGGATGGCAGTACTG 14 CDK2 4 TCTCCAAGTGAGACTGAGGGTAAGCAGAGAGATCTCTCGG 18 CDK2 5 TCTCCAAGTGAGACTGAGGG TTAAGCAGAGAGATCTCTCG19 CDK3 1 TGTTTCCCAGGCAGCTCTGT TCTCCGATCTTCTCTACCTT 19 CDK3 2TTTCCCAGGCAGCTCTGTGG TCTCCGATCTTCTCTACCTT 17 CDK3 3 TTCCCAGGCAGCTCTGTGGCTCTCCGATCTTCTCTACCTT 16 CDK3 4 TCCCAGGCAGCTCTGTGGCC TCTCCGATCTTCTCTACCTT15 CDK3 5 TGGATATGTTCCAGAAGGTA TACACCACCCCATAGGTGCC 15 CDK3 1TGCCCACGGCTGTGCCCTTG TGGCAGTGCTTGGGACCCCC 19 CDK3 2 TGTGCCCTTGTTTCTTGCAGTCCCTGATGGCAGTGCTTGG 16 CDK3 3 TTTCTTGCAGGGAGATGGAG TGAGCAGCGAGATCTCCCTG20 CDK3 4 TTCTTGCAGGGAGATGGAGG TGAGCAGCGAGATCTCCCTG 19 CDK3 5TTCTTGCAGGGAGATGGAGG TTGAGCAGCGAGATCTCCCT 20 CDK4 1 TGTGATTGTAGGGTCTCCCTTGGCTCATATCGAGAGGTAG 14 CDK4 2 TGATTGTAGGGTCTCCCTTG TCAGCCACTGGCTCATATCG20 CDK4 3 TTGTAGGGTCTCCCTTGATC TCAGCCACTGGCTCATATCG 17 CDK4 4TGTAGGGTCTCCCTTGATCT TCAGCCACTGGCTCATATCG 16 CDK4 5 TAGGGTCTCCCTTGATCTGATCAGCCACTGGCTCATATCG 14 CDK4 1 TTGAAAAGTGAGCATTTACT TCGGGATGTGGCACAGACGT16 CDK4 2 TTGAAAAGTGAGCATTTACT TTCGGGATGTGGCACAGACG 17 CDK4 3TGAAAAGTGAGCATTTACTC TCGGGATGTGGCACAGACGT 15 CDK4 4 TGAAAAGTGAGCATTTACTCTTCGGGATGTGGCACAGACG 16 CDK4 5 TGAAAAGTGAGCATTTACTC TCAGTTCGGGATGTGGCACA20 CDK5 1 TACGAGAAACTGGAAAAGAT TGCAGGAACATCTCGAGATT 15 CDK5 2TACGAGAAACTGGAAAAGAT TTGCAGGAACATCTCGAGAT 16 CDK5 3 TACGAGAAACTGGAAAAGATTCTTGCAGGAACATCTCGAG 18 CDK5 1 TCCTTCCCCTAGGCACCTAC TGAGTCTCCCGGTTTTTGGC15 CDK5 2 TCCTTCCCCTAGGCACCTAC TCATGAGTCTCCCGGTTTTT 18 CDK5 3TCCTTCCCCTAGGCACCTAC TCTCATGAGTCTCCCGGTTT 20 CDK5 4 TTCCCCTAGGCACCTACGGATCATGAGTCTCCCGGTTTTT 15 CDK5 5 TTCCCCTAGGCACCTACGGA TCTCATGAGTCTCCCGGTTT17 CDK6 1 TGTGCCGCGCTGACCAGCAG TAGGCGCCCTCCCCGATCTC 15 CDK6 2TGTGCCGCGCTGACCAGCAG TCCCATAGGCGCCCTCCCCG 20 CDK6 3 TGCCGCGCTGACCAGCAGTATCCCATAGGCGCCCTCCCCG 18 CDK6 4 TGCCGCGCTGACCAGCAGTA TTCCCATAGGCGCCCTCCCC19 CDK6 5 TGACCAGCAGTACGAATGCG TGAACACCTTCCCATAGGCG 19 CDK6 1TCTAGGTTGTTTGATGTGTG TAGTTTGGTTTCTCTGTCTG 14 CDK6 2 TCTAGGTTGTTTGATGTGTGTAAAGTTAGTTTGGTTTCTC 20 CDK6 3 TAGGTTGTTTGATGTGTGCA TAAAGTTAGTTTGGTTTCTC18 CDK6 4 TTGTTTGATGTGTGCACAGT TAAAGTTAGTTTGGTTTCTC 14 CDK6 5TTGATGTGTGCACAGTGTCA TCAAACACTAAAGTTAGTTT 18 EGFR 1 TCCGGGACGGCCGGGGCAGCTCGCCGGGCAGAGCGCAGCC 15 EGFR 1 TCTTCCAGTTTGCCAAGGCA TCAAAAGTGCCCAACTGCGT14 EGFR 2 TCTTCCAGTTTGCCAAGGCA TGATCTTCAAAAGTGCCCAA 20 EGFR 3TTCCAGTTTGCCAAGGCACG TGATCTTCAAAAGTGCCCAA 18 EGFR 4 TCCAGTTTGCCAAGGCACGATGATCTTCAAAAGTGCCCAA 17 EGFR 5 TCACGCAGTTGGGCACTTTT TGAACATCCTCTGGAGGCTG14 HIF1A 1 TGAAGACATCGCGGGGACCG TGTCGTTCGCGCCGCCGGCG 15 HIF1A 2TGAAGACATCGCGGGGACCG TTGTCGTTCGCGCCGCCGGC 16 HIF1A 3TGAAGACATCGCGGGGACCG TCTTGTCGTTCGCGCCGCCG 18 HIF1A 4TGAAGACATCGCGGGGACCG TTCTTGTCGTTCGCGCCGCC 19 HIF1A 5TGAAGACATCGCGGGGACCG TTTCTTGTCGTTCGCGCCGC 20 HIF1A 1TCTCGTGTTTTTCTTGTTGT TCTTTTCGACGTTCAGAACT 14 HIF1A 2TCTCGTGTTTTTCTTGTTGT TTCTTTTCGACGTTCAGAAC 15 HIF1A 3TCTCGTGTTTTTCTTGTTGT TTTCTTTTCGACGTTCAGAA 16 HIF1A 4TCTCGTGTTTTTCTTGTTGT TTTTCTTTTCGACGTTCAGA 17 HIF1A 5TTCTTGTTGTTGTTAAGTAG TCGAGACTTTTCTTTTCGAC 14 HSPA4 1TGGTGGGCATAGACCTGGGC TGCCGCCGGCGCGGGCCACA 20 HSPA4 2TGGGCATAGACCTGGGCTTC TGCCGCCGGCGCGGGCCACA 17 HSPA4 3TAGACCTGGGCTTCCAGAGC TCGATGCCGCCGGCGCGGGC 15 HSPA4 4TAGACCTGGGCTTCCAGAGC TCTCGATGCCGCCGGCGCGG 17 HSPA4 5TAGACCTGGGCTTCCAGAGC TAGTCTCGATGCCGCCGGCG 20 HSPA4 1TCTTAAGTGCTTTTTTTGTC TGAACGATTCTTAGGACCAA 20 HSPA4 2TTAAGTGCTTTTTTTGTCTT TGAACGATTCTTAGGACCAA 18 HSPA4 3TTAAGTGCTTTTTTTGTCTT TTGAACGATTCTTAGGACCA 19 HSPA4 4TAAGTGCTTTTTTTGTCTTC TGAACGATTCTTAGGACCAA 17 HSPA4 5TAAGTGCTTTTTTTGTCTTC TTGAACGATTCTTAGGACCA 18 HSP90AA1 1TGCCCCCGTGTTCGGGCGGG TCCCGAAGGGAGGGCCCAGG 15 HSP90AA1 2TGCCCCCGTGTTCGGGCGGG TGTCCCGAAGGGAGGGCCCA 17 HSP90AA1 3TCCTGGGCCCTCCCTTCGGG TCGCGCGGGTATTCAGCACT 20 HSP90AA1 4TGGGCCCTCCCTTCGGGACA TCGCGCGGGTATTCAGCACT 17 HSP90AA1 5TCCCTTCGGGACAGGGACTG TCCAGACGGTCGCGCGGGTA 19 HSP90AA1 1TCCAGAAGATTGTGTTTATG TCTTGGTACCAGTTAACAGG 14 HSP90AA1 2TGTGTTTATGTTCCCAGCAG TTGGGCCTTTTCTTGGTACC 14 HSP90AA1 3TCCCAGCAGGGCACCTGTTA TGCCAGAGAAACACTTGGGC 17 HSP90AA1 4TAACTGGTACCAAGAAAAGG TCCAGACACCATCAGATGCC 15 HSP90AA1 5TAACTGGTACCAAGAAAAGG TGGATCCAGACACCATCAGA 19 MYC 1 TCCAGCAGCCTCCCGCGACGTAGTTCCTGTTGGTGAAGCT 15 MYC 2 TCCAGCAGCCTCCCGCGACG TCATAGTTCCTGTTGGTGAA18 MYC 3 TCCCGCGACGATGCCCCTCA TCGAGGTCATAGTTCCTGTT 14 MYC 4TCCCGCGACGATGCCCCTCA TAGTCGAGGTCATAGTTCCT 17 MYC 5 TCCCGCGACGATGCCCCTCATCGTAGTCGAGGTCATAGTT 20 PKN3 1 TGCAGCCTGGGCCGAGCCAG TGGCCCGGCGGATCACCTCC20 PKN3 2 TGGGCCGAGCCAGTGGCCCC TGGATGGCCCGGCGGATCAC 17 PKN3 3TGGGCCGAGCCAGTGGCCCC TCTGGATGGCCCGGCGGATC 19 PKN3 4 TGGGCCGAGCCAGTGGCCCCTTCTGGATGGCCCGGCGGAT 20 PKN3 5 TGGCCCCCAGAGGATGAGAA TCAGCTCTTTCTGGATGGCC15 RRM2 1 TGGGAAGGGTCGGAGGCATG TGGCTTTGGTGCCCCGGCCC 16 RRM2 2TGGGAAGGGTCGGAGGCATG TTGGCTTTGGTGCCCCGGCC 17 RRM2 3 TCGGAGGCATGGCACAGCCATTCCCATTGGCTTTGGTGCC 14 RRM2 4 TGGCACAGCCAATGGGAAGG TCCCGGCCCTTCCCATTGGC14 RRM2 5 TGCACCCTGTCCCAGCCGTC TGGAGGCGCAGCGAAGCAGA 17 APC 1TATGTACGCCTCCCTGGGCT TGGTACAGAAGCGGGCAAAG 15 APC 2 TGTACGCCTCCCTGGGCTCGTGAGGGTGGTACAGAAGCGG 19 APC 3 TACGCCTCCCTGGGCTCGGG TGAGGGTGGTACAGAAGCGG17 APC 4 TCGGGTCCGGTCGCCCCTTT TCCAGGACCCGAGAACTGAG 18 APC 5TCCGGTCGCCCCTTTGCCCG TGCTCCAGGACCCGAGAACT 16 APC 1 TTAAACAACTACAAGGAAGTTCAATCTGTCCAGAAGAAGC 18 APC 2 TAAACAACTACAAGGAAGTA TCAATCTGTCCAGAAGAAGC17 APC 3 TACAAGGAAGTATTGAAGAT TAATAAATCAATCTGTCCAG 16 APC 4TATTGAAGATGAAGCTATGG TAAGACGCTCTAATAAATCA 16 APC 5 TATTGAAGATGAAGCTATGGTTAAGACGCTCTAATAAATC 17 BRCA1 1 TGGATTTATCTGCTCTTCGCTGCATAGCATTAATGACATT 15 BRCA1 2 TGGATTTATCTGCTCTTCGCTCTGCATAGCATTAATGACA 17 BRCA1 3 TTATCTGCTCTTCGCGTTGATAAGATTTTCTGCATAGCAT 20 BRCA1 4 TATCTGCTCTTCGCGTTGAATAAGATTTTCTGCATAGCAT 19 BRCA1 5 TCTGCTCTTCGCGTTGAAGATAAGATTTTCTGCATAGCAT 17 BRCA1 1 TGCTAGTCTGGAGTTGATCATGCAAAATATGTGGTCACAC 19 BRCA1 2 TGCTAGTCTGGAGTTGATCATTGCAAAATATGTGGTCACA 20 BRCA1 3 TAGTCTGGAGTTGATCAAGGTGCAAAATATGTGGTCACAC 16 BRCA1 4 TAGTCTGGAGTTGATCAAGGTTGCAAAATATGTGGTCACA 17 BRCA1 5 TAGTCTGGAGTTGATCAAGGTACTTGCAAAATATGTGGTC 20 BRCA2 1 TGCCTATTGGATCCAAAGAGTGCAGCGTGTCTTAAAAATT 17 BRCA2 2 TGCCTATTGGATCCAAAGAGTTGCAGCGTGTCTTAAAAAT 18 BRCA2 3 TGCCTATTGGATCCAAAGAGTGTTGCAGCGTGTCTTAAAA 20 BRCA2 4 TATTGGATCCAAAGAGAGGCTTGCAGCGTGTCTTAAAAAT 14 BRCA2 5 TATTGGATCCAAAGAGAGGCTGTTGCAGCGTGTCTTAAAA 16 BRCA2 1 TAGATTTAGGACCAATAAGTTGGAGCTTCTGAAGAAAGTT 16 BRCA2 2 TTAGGACCAATAAGTCTTAATAGGGTGGAGCTTCTGAAGA 16 BRCA2 3 TTAGGACCAATAAGTCTTAATATAGGGTGGAGCTTCTGAA 18 BRCA2 4 TTAGGACCAATAAGTCTTAATTATAGGGTGGAGCTTCTGA 19 BRCA2 5 TAGGACCAATAAGTCTTAATTATAGGGTGGAGCTTCTGAA 17 TP53 1 TCACTGCCATGGAGGAGCCG TGACTCAGAGGGGGCTCGAC15 TP53 2 TCACTGCCATGGAGGAGCCG TCCTGACTCAGAGGGGGCTC 18 TP53 3TCACTGCCATGGAGGAGCCG TTCCTGACTCAGAGGGGGCT 19 TP53 4 TCACTGCCATGGAGGAGCCGTTTCCTGACTCAGAGGGGGC 20 TP53 5 TGCCATGGAGGAGCCGCAGT TCCTGACTCAGAGGGGGCTC14 APP 1 TTCTTTCAGGTACCCACTGA TGGCAATCTGGGGTTCAGCC 18 APP 2TCTTTCAGGTACCCACTGAT TGGCAATCTGGGGTTCAGCC 17 APP 3 TTTCAGGTACCCACTGATGGTGGCAATCTGGGGTTCAGCC 15 APP 4 TTCAGGTACCCACTGATGGT TGGCAATCTGGGGTTCAGCC14 APP 5 TACCCACTGATGGTAATGCT TGCCACAGAACATGGCAATC 20 IAPP 1TGGGCATCCTGAAGCTGCAA TGGTTCAATGCAACAGAGAG 15 IAPP 2 TGGGCATCCTGAAGCTGCAATCAGATGGTTCAATGCAACA 20 IAPP 3 TGCAAGTATTTCTCATTGTG TGGGTGTAGCTTTCAGATGG17 IAPP 4 TGCTCTCTGTTGCATTGAAC TTACCAACCTTTCAATGGGT 14 IAPP 1TGTTACCAGTCATCAGGTGG TGCGTTGCACATGTGGCAGT 17 IAPP 2 TTACCAGTCATCAGGTGGAATGCGTTGCACATGTGGCAGT 15 IAPP 3 TACCAGTCATCAGGTGGAAA TGCGTTGCACATGTGGCAGT14 IAPP 4 TCATCAGGTGGAAAAGCGGA TGCCAGGCGCTGCGTTGCAC 18 IAPP 5TCATCAGGTGGAAAAGCGGA TTGCCAGGCGCTGCGTTGCA 19 SNCA 1 TTTTGTAGGCTCCAAAACCATTACCTGTTGCCACACCATG 14 SNCA 2 TTTTGTAGGCTCCAAAACCA TGGAGCTTACCTGTTGCCAC20 SNCA 3 TTTGTAGGCTCCAAAACCAA TGGAGCTTACCTGTTGCCAC 19 SNCA 4TTGTAGGCTCCAAAACCAAG TGGAGCTTACCTGTTGCCAC 18 SNCA 5 TGTAGGCTCCAAAACCAAGGTGGAGCTTACCTGTTGCCAC 17 SOD1 1 TAGCGAGTTATGGCGACGAA TGCACTGGGCCGTCGCCCTT16 SOD1 2 TTATGGCGACGAAGGCCGTG TGCCCTGCACTGGGCCGTCG 14 SOD1 3TTATGGCGACGAAGGCCGTG TGATGCCCTGCACTGGGCCG 17 SOD1 4 TTATGGCGACGAAGGCCGTGTGATGATGCCCTGCACTGGG 20 SOD1 5 TATGGCGACGAAGGCCGTGT TGATGCCCTGCACTGGGCCG16 SOD1 1 TAATGGACCAGTGAAGGTGT TGCAGGCCTTCAGTCAGTCC 14 SOD1 2TAATGGACCAGTGAAGGTGT TCCATGCAGGCCTTCAGTCA 18 SOD1 3 TGGACCAGTGAAGGTGTGGGTCCATGCAGGCCTTCAGTCA 15 SOD1 4 TGGACCAGTGAAGGTGTGGG TGGAATCCATGCAGGCCTTC20 SOD1 5 TGTGGGGAAGCATTAAAGGA TCATGAACATGGAATCCATG 15

In some embodiments, the target DNA molecule comprises a gene that isoverexpressed in cancer. Example genes that are overexpressed in cancerinclude, but are not limited to: ABL1, BIRC5, BLK, BTK, CDK familymembers, EGFR, ERBB2, FAS, FGR, FLT4, FRK, FYN, HCK, HIF1A, HRAS,HSP90AA1, HSP90AA1, HSPA4, KDR, KIF11, KIF11, KIF20A, KIF21A, KIF25,KIT, KRAS, LCK, LYN, MAPK1, MET, MYC, MYH1, MYO1G, NRAS, NTRK1, PDGFB,PDGFRA, PDGFRB, PKN3, PLK1, RAF1, RB1, RET, RRM1, RRM2, SRC, TNF, TPM2,TYRO3, VEGFA, VEGFB, VEGFC, YES1, and ZAP70. In some embodiments, thetarget DNA molecule comprises a gene selected from: ABL1, BIRC5, BLK,BTK, a CDK family member, EGFR, ERBB2, FAS, FGR, FLT4, FRK, FYN, HCK,HIF1A, HRAS, HSP90AA1, HSP90AA1, HSPA4, KDR, KIF11, KIF11, KIF20A,KIF21A, KIF25, KIT, KRAS, LCK, LYN, MAPK1, MET, MYC, MYH1, MYO1G, NRAS,NTRK1, PDGFB, PDGFRA, PDGFRB, PKN3, PLK1, RAF1, RB1, RET, RRM1, RRM2,SRC, TNF, TPM2, TYRO3, VEGFA, VEGFB, VEGFC, YES1, and ZAP70 or afragment or variant thereof. In other embodiments, the target DNAmolecule comprises a gene that is mutated in cancer. Example genes thatare mutated in cancer include, but are not limited to: AIM1, APC, BRCA1,BRCA2, CDKN1B, CDKN2A, FAS, FZD family members, HNF1A, HOPX, KLF6, MEN1,MLH1, NTRK1, PTEN, RARRES1, RB1, SDHB, SDHD, SFRP1, ST family members,TNF, TP53, TP63, TP73, VBP1, VHL, WNT family members, BRAF, CTNNB1,PIK3CA, PIK3R1, SMAD4, and YPEL3. In some embodiments, the target DNAmolecule comprises a gene selected from: AIM1, APC, BRCA1, BRCA2,CDKN1B, CDKN2A, FAS, a FZD family member, HNF1A, HOPX, KLF6, MEN1, MLH1,NTRK1, PTEN, RARRES1, RB1, SDHB, SDHD, SFRP1, a ST family member, TNF,TP53, TP63, TP73, VBP1, VHL, a WNT family member, BRAF, CTNNB1, PIK3CA,PIK3R1, SMAD4, and YPEL3 or a fragment or variant thereof. In oneembodiment, the method further comprises administering to a patient atherapeutically effective amount of a repair template.

Mutations in certain genes can increase the likelihood of a cellbecoming cancerous. In certain situations, however, it can bedetrimental to inactivate a cancer-associated gene in non-cancerouscells, for example, if the non-mutated form of the cancer-associatedgene is beneficial. It has now been discovered that gene-editingproteins can be used to specifically inactivate, partially orcompletely, mutated forms of genes. Examples of cancer-associatedmutations include, but are not limited to: ALK (F1174, R1275), APC(R876, Q1378, R1450), BRAF (V600), CDKN2A (R58, R80, H83, D84, E88,D108G, W110, P114), CTNNB1 (D32, S33, G34, S37, T41, or S45), EGFR(G719, T790, L858), EZH2 (Y646), FGFR3 (S249, Y373), FLT3 (D835), GNAS(R201), HRAS (G12, G13, Q61), IDH1 (R132), JAK2 (V617), KIT (D816), KRAS(G12, G13), NRAS (G12, G13, Q61), PDGFRA (D842), PIK3CA (E542, E545,H1047), PTEN (R130), and TP53 (R175, H179, G245, R248, 8249, 8273,W282). Certain embodiments are therefore directed to a gene-editingprotein that binds to a disease-associated mutation. In one embodiment,the gene-editing protein binds to DNA containing a specific mutationwith greater affinity than DNA that does not contain the mutation. Inanother embodiment, the disease is cancer.

Neurodegenerative diseases, including Alzheimer's disease, Parkinson'sdisease, and dementia with Lewy bodies, are characterized by theprogressive loss of function and/or death of cells of the central and/orperipheral nervous systems. Disease progression can be accompanied bythe accumulation of protein-rich plaques that can comprise the proteinα-synuclein (encoded, in humans, by the SNCA gene). As a result,researchers have sought to develop therapeutics that can break up theseplaques, for example, by means of an antibody that binds to the plaqueand tags it for destruction by the immune system. However, in manycases, breaking up plaques has little or no effect on patient symptomsor the progression of the disease. It has now been discovered that thefailure of existing therapies that target neurodegenerativedisease-associated plaques is due in part to the inability of thenervous system to repair the damage to cells that occurs during theearly stages of plaque formation. It has been further discovered thatinducing a cell to express one or more gene-editing proteins that targetthe SNCA gene can result in disruption of the SNCA gene, can induce thecell to express a plaque-resistant variant of α-synuclein protein, canslow or halt the growth of neurodegenerative disease-associated plaques,can protect the cell and nearby cells from the damaging effects ofneurodegenerative disease-associated plaques, can slow and/or halt theprogression of neurodegenerative diseases, including Alzheimer'sdisease, Parkinson's disease, and dementia with Lewy bodies, and canresult in a reduction of symptoms and/or gain of function in patientswith neurodegenerative diseases, including Alzheimer's disease,Parkinson's disease, and dementia with Lewy bodies. Otherneurodegenerative diseases include, for example, vision loss, includingblindness, hearing loss, including deafness, balance disorders, loss oftaste and/or smell, and other sensory disorders. Certain embodiments aretherefore directed to a gene-editing protein that targets the SNCA gene.In one embodiment, the gene-editing protein binds to one or more regionsin the SNCA gene. In another embodiment, the gene-editing protein bindsto one or more nucleic-acid sequences that encode SEQ ID NO: 51 or abiologically active fragment, variant or analogue thereof. Otherembodiments are directed to a method for treating a neurodegenerativedisease comprising administering to a patient a therapeuticallyeffective amount of a gene-editing protein or a nucleic acid encoding agene-editing protein, wherein the gene-editing protein is capable ofbinding to a nucleotide sequence that encodes a protein that formsdisease-associated plaques, and resulting in a reduction ofdisease-associated plaques in the patient and/or delayed or haltedprogression of the disease. In one embodiment, the nucleotide sequencecomprises the SNCA gene. In another embodiment, the nucleotide sequenceencodes α-synuclein. In a further embodiment, the neurodegenerativedisease is selected from: Parkinson's disease, Alzheimer's disease, anddementia.

Certain embodiments are directed to a method for identifying adisease-causing toxicant comprising transfecting a cell with agene-editing protein or a nucleic acid encoding a gene-editing proteinto alter the DNA sequence of the cell, wherein the altered DNA sequenceconfers susceptibility to a disease, contacting the cell with asuspected disease-causing toxicant, and assessing the degree to whichthe cell exhibits a phenotype associated with the disease. In oneembodiment, the disease is a neurodegenerative disease, autoimmunedisease, respiratory disease, reproductive disorder or cancer. Otherembodiments are directed to a method for assessing the safety of atherapeutic substance comprising transfecting a cell with a gene-editingprotein or a nucleic acid encoding a gene-editing protein to alter theDNA sequence of the cell, wherein the altered DNA sequence conferssusceptibility to one or more toxic effects of the therapeuticsubstance, contacting the cell with the therapeutic substance, andmeasuring one or more toxic effects of the therapeutic substance on thecell. Still other embodiments are directed to a method for assessing theeffectiveness of a therapeutic substance comprising transfecting a cellwith a gene-editing protein or a nucleic acid encoding a gene-editingprotein to alter the DNA sequence of the cell, wherein the altered DNAsequence causes the cell to exhibit one or more disease-associatedphenotypes, contacting the cell with the therapeutic substance, andmeasuring the degree to which the one or more disease-associatedphenotypes are reduced.

In some embodiments, the patient is diagnosed with a proteopathy.Example proteopathies and proteopathy-associated genes are given inTable 6, and are included by way of example, and not by way oflimitation. In one embodiment, the proteopathy is selected from: AA(secondary) amyloidosis, Alexander disease, Alzheimer's disease,amyotrophic lateral sclerosis, aortic medial amyloidosis, ApoAIamyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, bibrinogenamyloidosis, cardiac atrial amyloidosis, cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy, cerebralβ-amyloid angiopathy, dialysis amyloidosis, familial amyloidcardiomyopathy, familial amyloid polyneuropathy, familial amyloidosis(Finnish type), familial British dementia, familial Danish dementia,frontotemporal lobar degeneration, hereditary cerebral amyloidangiopathy, hereditary lattice corneal dystrophy, Huntington's disease,inclusion body myositis/myopathy, lysozyme amyloidosis, medullarythyroid carcinoma, odontogenic (Pindborg) tumor amyloid, Parkinson'sdisease, pituitary prolactinoma, prion diseases, pulmonary alveolarproteinosis, retinal ganglion cell degeneration in glaucoma, retinitispigmentosa with rhodopsin mutations, senile systemic amyloidosis,serpinopathies, synucleinopathies, tauopathies, type II diabetes,dementia pugilistica (chronic traumatic encephalopathy), frontotemporaldementia, frontotemporal lobar degeneration, gangliocytoma,ganglioglioma, Hallervorden-Spatz disease, lead encephalopathy,lipofuscinosis, Lytico-Bodig disease, meningioangiomatosis, progressivesupranuclear palsy, subacute sclerosing panencephalitis,tangle-predominant dementia, and tuberous sclerosis. In anotherembodiment, the target DNA molecule comprises a gene selected from:APOA1, APOA2, APOA4, APP, B2M, CALCA, CST3, FGA, FGB, FGG, FUS, GFAP,GSN, HTT, IAPP, ITM2B, LYZ, MAPT, MFGE8, NOTCH3, NPPA, ODAM, PRL, PRNP,RHO, a SAA family member, a SERPIN family member, SFTPC, SNCA, a SODfamily member, TARDBP, TGFBI, and TRR or a fragment or variant thereof.In a further embodiment, the target DNA molecule encodes a gene selectedfrom Table 6 or a fragment thereof, and the patient is diagnosed withthe corresponding disease listed in Table 6.

TABLE 6 Exemplary Proteopathies and Proteopathy-Associated GenesGene/Family Disease/Condition APOA1 ApoAI amyloidosis APOA2 ApoAIIamyloidosis APOA4 ApoAIV amyloidosis APP Cerebral β-amyloid angiopathyAPP Retinal ganglion cell degeneration in glaucoma APP Inclusion bodymyositis/myopathy APP, MAPT Alzheimer's disease B2M Dialysis amyloidosisCALCA Medullary thyroid carcinoma CST3 Hereditary cerebral amyloidangiopathy (Icelandic) FGA, FGB, FGG Fibrinogen amyloidosis GFAPAlexander disease GSN Familial amyloidosis, Finnish type HTTHuntington's disease IAPP Type II diabetes ITM2B Familial Britishdementia ITM2B Familial Danish dementia LYZ Lysozyme amyloidosis MAPTTauopathies (multiple) MFGE8 Aortic medial amyloidosis NOTCH3 Cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL) NPPA Cardiac atrial amyloidosis ODAMOdontogenic (Pindborg) tumor amyloid PRL Pituitary prolactinoma PRNPPrion diseases (multiple) RHO Retinitis pigmentosa with rhodopsinmutations SAA family genes AA (secondary) amyloidosis SERPIN familygenes Serpinopathies (multiple) SFTPC Pulmonary alveolar proteinosisSNCA Parkinson's disease and other synucleinopathies (multiple) SNCAOther synucleinopathies SOD family genes, Amyotrophic lateral sclerosis(ALS) TARDBP, FUS TARDBP, FUS Frontotemporal lobar degeneration (FTLD)TGFBI Hereditary lattice corneal dystrophy LMNA Hutchinson-GilfordProgeria Syndrome TRR Senile systemic amyloidosis (SSA), familialamyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC)

Example tauopathies include, but are not limited to Alzheimer's disease,Parkinson's disease, and Huntington's disease. Other example tauopathiesinclude: dementia pugilistica (chronic traumatic encephalopathy),frontotemporal dementia, frontotemporal lobar degeneration,gangliocytoma, ganglioglioma, Hallervorden-Spatz disease, leadencephalopathy, lipofuscinosis, Lytico-Bodig disease,meningioangiomatosis, progressive supranuclear palsy, subacutesclerosing panencephalitis, tangle-predominant dementia, and tuberoussclerosis. In some embodiments, the patient is diagnosed with atauopathy. In one embodiment, the tauopathy is selected from Alzheimer'sdisease, Parkinson's disease, and Huntington's disease. In anotherembodiment, the tauopathy is selected from: dementia pugilistica(chronic traumatic encephalopathy), frontotemporal dementia,frontotemporal lobar degeneration, gangliocytoma, ganglioglioma,Hallervorden-Spatz disease, lead encephalopathy, lipofuscinosis,Lytico-Bodig disease, meningioangiomatosis, progressive supranuclearpalsy, subacute sclerosing panencephalitis, tangle-predominant dementia,and tuberous sclerosis.

Autoimmune diseases, including but not limited to lupus, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), and transplantrejection, are characterized by symptoms caused in part by one or moreelements of the immune system attacking uninfected and non-cancerousisogenic cells and/or tissues. Certain embodiments are thereforedirected to a method for treating an autoimmune disease. In oneembodiment, the autoimmune disease is selected from: lupus, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), and transplantrejection. In another embodiment, the target DNA molecule encodes apolypeptide sequence that can be recognized by the host immune system.

Infectious agents can contain nucleic acid sequences that are notpresent in the host organism. It has now been discovered thatgene-editing proteins can be used to eliminate, reduce or otherwisealter, in whole or in part, infectious agents and/or the effects ofinfection, and that when used in this manner, gene-editing proteins andnucleic acids encoding gene-editing proteins, can constitute potentanti-infection therapeutics. Infectious agents that can be treated insuch a manner include, but are not limited to: viruses, bacteria, fungi,yeast, and parasites. Certain embodiments are therefore directed to amethod for inducing a cell to express a gene-editing protein thattargets one or more infectious agent-associated sequences. In oneembodiment, the cell is one of: a bacterial cell, a fungal cell, a yeastcell, and a parasite cell. In another embodiment, the cell is amammalian cell. In a further embodiment, the cell is a human cell. Otherembodiments are directed to a therapeutic composition comprising anucleic acid that encodes one or more gene-editing proteins that targetsone or more infectious agent-associated sequences. Certain embodimentsare directed to a method for inducing a cell to express a gene-editingprotein that targets one or more sequences associated withsusceptibility or resistance to infection. Other embodiments aredirected to a therapeutic composition comprising a nucleic acid thatencodes one or more gene-editing proteins that targets one or moresequences associated with susceptibility or resistance to infection. Inone embodiment, the cell is transfected with a nucleic acid encoding oneor more gene-editing proteins and a nucleic acid encoding one or morerepair templates. In another embodiment, the repair template contains aresistance gene or a biologically active fragment or variant thereof. Ina further embodiment, the repair template contains an RNAi sequence. Ina still further embodiment, the RNAi sequence is a shRNA. Otherembodiments are directed to a method for treating an infectious diseasecomprising administering to a patient a therapeutically effective amountof a gene-editing protein or a nucleic acid encoding a gene-editingprotein, wherein the gene-editing protein is capable of binding to oneor more nucleotide sequences that are present in the infectious agent.

It has now been discovered that the ratio of non-homologous end joiningevents to homologous recombination events can be altered by altering theexpression and/or function of one or more components of a DNA-repairpathway. Non-limiting examples of genes that encode components of aDNA-repair pathway include, but are not limited to: Artemis, BLM, CtIP,DNA-PK, DNA-PKcs, EXO1, FEN1, Ku70, Ku86, LIGIII, LIGIV, MRE11, NBS1,PARP1, RAD50, RAD54B, XLF, XRCC1, XRCC3, and XRCC4. Certain embodimentsare therefore directed to a method for altering the expression and/orfunction of one or more components of a DNA-repair pathway. In certainembodiments, the expression and/or function is increased. In otherembodiments, the expression and/or function is decreased. DNA-dependentprotein kinase (DNA-PK) is a component of the non-homologous end-joiningDNA-repair pathway. It has now been discovered that repair viahomologous recombination can be increased by altering the expression ofDNA-PK. In one embodiment, a cell is contacted with a DNA-PK inhibitor.Example DNA-PK inhibitors include, but are not limited to: Compound 401(2-(4-Morpholinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one), DMNB, IC87361,LY294002, NU7026, NU7441, OK-1035, PI 103 hydrochloride, vanillin, andwortmannin.

Genetic mutations can affect the length of a protein product, forexample, by introducing a stop codon and/or disrupting an open readingframe. Certain diseases, including Duchenne muscular dystrophy, can becaused by the production of truncated and/or frameshifted proteins. Ithas now been discovered that gene-editing proteins can be used to treatdiseases that are associated with the production of one or moretruncated and/or frameshifted proteins. In one embodiment, thegene-editing protein creates a double strand break within about 1 kb orabout 0.5 kb or about 0.1 kb of an exon containing adisease-contributing mutation. In another embodiment, the gene-editingprotein is co-expressed with a DNA sequence comprising one or morewild-type sequences. In certain embodiments, the DNA is single-stranded.In other embodiments, the DNA is double-stranded. Diseases caused by theexpression of truncated proteins can be treated by exon skipping. It hasnow been discovered that gene-editing proteins can be used to induceexon skipping. In one embodiment, the gene-editing protein creates adouble-strand break within about 1 kb or about 0.5 kb or about 0.1 kb ofthe exon to be skipped. In another embodiment, the gene-editing proteincreates a double-strand break within about 1 kb or about 0.5 kb or about0.1 kb of an intron upstream of the exon to be skipped. In anotherembodiment, the gene-editing protein creates a double-strand breakwithin about 1 kb or about 0.5 kb or about 0.1 kb of the splice-acceptorsite of an intron upstream of the exon to be skipped.

Nucleic acids, including liposomal formulations containing nucleicacids, when delivered in vivo, can accumulate in the liver and/orspleen. It has now been discovered that nucleic acids encodinggene-editing proteins can modulate gene expression in the liver andspleen, and that nucleic acids used in this manner can constitute potenttherapeutics for the treatment of liver and spleen diseases. Certainembodiments are therefore directed to a method for treating liver and/orspleen disease by delivering to a patient a nucleic acid encoding one ormore gene-editing proteins. Other embodiments are directed to atherapeutic composition comprising a nucleic acid encoding one or moregene-editing proteins, for the treatment of liver and/or spleen disease.Diseases and conditions of the liver and/or spleen that can be treatedinclude, but are not limited to: hepatitis, alcohol-induced liverdisease, drug-induced liver disease, Epstein Barr virus infection,adenovirus infection, cytomegalovirus infection, toxoplasmosis, RockyMountain spotted fever, non-alcoholic fatty liver disease,hemochromatosis, Wilson's Disease, Gilbert's Disease, and cancer of theliver and/or spleen. Other examples of sequences (including genes, genefamilies, and loci) that can be targeted by gene-editing proteins usingthe methods of the present invention are set forth in Table 7, and aregiven by way of example, and not by way of limitation.

TABLE 7 Exemplary Gene Editing-Protein Targets Disease/ConditionGene/Family/Locus Age-related macular VEGF family degenerationAlzheimer's disease APP, PSEN1, PSEN2, APOE, CR1, CLU, PICALM, BIN1,MS4A4, MS4A6E, CD2AP, CD33, EPHA1 Amyotrophic lateral SOD1 sclerosisCancer BRCA1, EGFR, MYC family, TP53, PKN3, RAS family, BIRC5, PTEN,RET, KIT, MET, APC, RBI, BRCA2, VEGF family, TNF, HNPCC1, HNPCC2, HNPCC5Cystic fibrosis CFTR Diabetes GCK, HNF1A, HNF4A, HNF1B Duchenne muscularDMD dystrophy Fanconi anemia BRCA2, FANCA, FANCB, FANCC, FANCD2, FANCE,FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP, RAD51CHemochromatosis HFE, HJV, HAMP, TFR2, SLC40A1 Hemophilia F8, F9, F11HIV/AIDS CCR5, CXCR4 Huntington's disease HTT Marfan's syndrome FBN1Neurofibromatosis NF1, NF2 Parkinson's disease SNCA, PRKN, LRRK2, PINK1,PARK7, ATP13A2 Safe-harbor locus in AAVS1 humans Safe-harbor locus inRosa26 mice and rats Sickle-cell anemia HBB Tay-Sachs disease HEXAXeroderma XPA, XPB, XPC, XPD, pigmentosum DDB2, ERCC4, ERCC5, ERCC6,RAD2, POLH Psoriasis, Rheumatoid TNF arthritis, Ankylosing spondylitis,Crohn's disease, Hidradenitis suppurativa, Refractory asthma Psoriasis,Rheumatoid JAK family arthritis, Polycythemia vera, Essentialthrombocythemia, Myeloid metaplasia with myelofibrosis

Certain embodiments are directed to a combination therapy comprising oneor more of the therapeutic compositions of the present invention and oneor more adjuvant therapies. Example adjuvant therapies are set forth inTable 8 and Table 5 of U.S. Provisional Application No. 61/721,302, thecontents of which are hereby incorporated by reference, and are given byway of example, and not by way of limitation.

TABLE 8 Exemplary Adjuvant Therapies Therapy Class Disease/ConditionExample Therapy Acetylcholinesterase inhibitors Myasthenia gravis,Glaucoma, Alzheimer's Edrophonium disease, Lewy body dementia, Posturaltachycardia syndrome Angiotensin-converting-enzyme Hypertension,Congestive heart failure Perindopril inhibitor Alkylating agents CancerCisplatin Angiogenesis inhibitors Cancer, Macular degenerationBevacizumab Angiotensin II receptor Hypertension, Diabetic nephropathy,Valsartan antagonists Congestive heart failure Antibiotics Bacterialinfection Amoxicillin Antidiabetic drugs Diabetes MetforminAntimetabolites Cancer, Infection 5-fluorouracil (5FU) Antisenseoligonucleotides Cancer, Diabetes, Amyotrophic lateral Mipomersensclerosis (ALS), Hypercholesterolemia Cytotoxic antibiotics CancerDoxorubicin Deep-brain stimulation Chronic pain, Parkinson's disease,Tremor, N/A Dystonia Dopamine agonists Parkinson's disease, Type IIdiabetes, Bromocriptine Pituitary tumors Entry/Fusion inhibitorsHIV/AIDS Maraviroc Glucagon-like peptide-1 agonists Diabetes ExenatideGlucocorticoids Asthma, Adrenal insufficiency, DexamethasoneInflammatory diseases, Immune diseases, Bacterial meningitisImmunosuppressive drugs Organ transplantation, Inflammatory Azathioprinediseases, Immune diseases Insulin/Insulin analogs Diabetes NPH insulinIntegrase inhibitors HIV/AIDS Raltegravir MAO-B inhibitors Parkinson'sdisease, Depression, Dementia Selegiline Maturation inhibitors HIV/AIDSBevirimat Nucleoside analog reverse- HIV/AIDS, Hepatitis B Lamivudinetranscriptase inhibitors Nucleotide analog reverse- HIV/AIDS, HepatitisB Tenofovir transcriptase inhibitors Non-nucleoside reverse- HIV/AIDSRilpivirine transcriptase inhibitors Pegylated interferon Hepatitis B/C,Multiple sclerosis Interferon beta-1a Plant alkaloids/terpenoids CancerPaclitaxel Protease inhibitors HIV/AIDS, Hepatitis C, Other viralTelaprevir infections Radiotherapy Cancer Brachytherapy Renin inhibitorsHypertension Aliskiren Statins Hypercholesterolemia AtorvastatinTopoisomerase inhibitors Cancer Topotecan Vasopressin receptorantagonist Hyponatremia, Kidney disease Tolvaptan

Pharmaceutical preparations may additionally comprise delivery reagents(a.k.a. “transfection reagents”) and/or excipients. Pharmaceuticallyacceptable delivery reagents, excipients, and methods of preparation anduse thereof, including methods for preparing and administeringpharmaceutical preparations to patients (a.k.a. “subjects”) are wellknown in the art, and are set forth in numerous publications, including,for example, in US Patent Appl. Pub. No. US 2008/0213377, the entiretyof which is hereby incorporated by reference.

For example, the present compositions can be in the formpharmaceutically acceptable salts. Such salts include those listed in,for example, J. Pharma. Sci. 66, 2-19 (1977) and The Handbook ofPharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are herebyincorporated by reference in their entirety. Non-limiting examples ofpharmaceutically acceptable salts include: sulfate, citrate, acetate,oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate,phenylacetate, trifluoroacetate, acrylate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate,α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate,caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate,malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate,phthalate, teraphthalate, propiolate, propionate, phenylpropionate,sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate,ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate,naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, tartarate salts, hydroxidesof alkali metals such as sodium, potassium, and lithium; hydroxides ofalkaline earth metal such as calcium and magnesium; hydroxides of othermetals, such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines,dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine;diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-loweralkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine,N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

The present pharmaceutical compositions can comprises excipients,including liquids such as water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The pharmaceutical excipients canbe, for example, saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea and the like. In addition, auxiliary,stabilizing, thickening, lubricating, and coloring agents can be used.In one embodiment, the pharmaceutically acceptable excipients aresterile when administered to a subject. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In various embodiments, the compositions described herein canadministered in an effective dose of, for example, from about 1 mg/kg toabout 100 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5 mg/kg toabout 25 mg/kg. The precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, and type of disease. Dosages can be readilyascertained by those of ordinary skill in the art from this disclosureand the knowledge in the art. For example, doses may be determined withreference Physicians' Desk Reference, 66th Edition, PDR Network; 2012Edition (Dec. 27, 2011), the contents of which are incorporated byreference in its entirety.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal, or buccal. Alternatively, administration may be by intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection,or by direct injection into cancer tissue. The agents disclosed hereinmay also be administered by catheter systems. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions asdescribed herein.

Upon formulation, solutions may be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations may easily be administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution generally is suitably buffered and the liquid diluent firstrendered isotonic with, for example, sufficient saline or glucose. Suchaqueous solutions may be used, for example, for intravenous,intramuscular, subcutaneous and intraperitoneal administration.Preferably, sterile aqueous media are employed as is known to those ofskill in the art, particularly in light of the present disclosure.

Exemplary subjects or patients refers to any vertebrate including,without limitation, humans and other primates (e.g., chimpanzees andother apes and monkey species), farm animals (e.g., cattle, sheep, pigs,goats, and horses), domestic mammals (e.g., dogs and cats), laboratoryanimals (e.g., rodents such as mice, rats, and guinea pigs), and birds(e.g., domestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like). In some embodiments,the subject is a mammal. In some embodiments, the subject is a human.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 RNA Synthesis

RNA encoding the human proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), andLin28 or TALENs targeting the human genes XPA, CCR5, TERT, MYC, andBIRC5, and comprising various combinations of canonical andnon-canonical nucleotides, was synthesized from DNA templates using theT7 High Yield RNA Synthesis Kit and the Vaccinia Capping System kit withmRNA Cap 2′-O-Methyltransferase (all from New England Biolabs, Inc.),according to the manufacturer's instructions and the present inventors'previously disclosed inventions (U.S. application Ser. No. 13/465,490(now U.S. Pat. No. 8,497,124), U.S. Provisional Application No.61/637,570, U.S. Provisional Application No. 61/664,494, InternationalApplication No. PCT/US12/67966, U.S. Provisional Application No.61/785,404, U.S. application Ser. No. 13/931,251, and U.S. ProvisionalApplication No. 61/842,874, the contents of all of which are herebyincorporated by reference in their entirety) (Table 9, FIG. 1A, FIG. 1B,and FIG. 15). The RNA was then diluted with nuclease-free water tobetween 100 ng/μL and 200 ng/μL. For certain experiments, an RNaseinhibitor (Superase.In, Life Technologies Corporation) was added at aconcentration of 1 μL/100 μg of RNA. RNA solutions were stored at 4° C.For reprogramming experiments, RNA encoding Oct4, Sox2, Klf4, c-Myc-2(T58A), and Lin28 was mixed at a molar ratio of 3:1:1:1:1.

TABLE 9 RNA Synthesis Reaction ivT Template Nucleotides Volume/μLYield/μg Oct4 A, G, U, C 10 64.9 Oct4 A, G, 0.25 4sU, C 10 64.3 Oct4 A,G, 0.5 4sU, C 10 62.8 Oct4 A, G, 0.75 4sU, C 10 51.9 Oct4 A, G, 4sU, C10 0 Oct4 A, 0.5 7dG, 0.75 4sU, 0.25 piC 20 70.1 Sox2 A, 0.5 7dG, 0.754sU, 0.25 piC 10 29.6 Klf4 A, 0.5 7dG, 0.75 4sU, 0.25 piC 10 29.5c-Myc-2 (T58A) A, 0.5 7dG, 0.75 4sU, 0.25 piC 10 25.9 Lin28 A, 0.5 7dG,0.75 4sU, 0.25 piC 10 36.7 Oct4 A, 0.5 7dG, 0.75 4sU, 0.5 piC 20 51.7Sox2 A, 0.5 7dG, 0.75 4sU, 0.5 piC 10 23.0 Klf4 A, 0.5 7dG, 0.75 4sU,0.5 piC 10 22.3 c-Myc-2 (T58A) A, 0.5 7dG, 0.75 4sU, 0.5 piC 10 21.4Lin28 A, 0.5 7dG, 0.75 4sU, 0.5 piC 10 23.3 Oct4 A, 0.5 7dG, 0.8 4sU,0.2 5mU, 0.5 piC 20 50.8 Oct4 A, 0.5 7dG, 0.7 4sU, 0.3 5mU, 0.5 piC 2058.3 Oct4 A, 0.5 7dG, 0.6 4sU, 0.4 5mU, 0.5 piC 20 58.3 Oct4 A, 0.5 7dG,0.5 4sU, 0.5 5mU, 0.5 piC 20 68.2 Oct4 A, 0.5 7dG, 0.4 4sU, 0.6 5mU, 0.5piC 20 78.7 Oct4 A, G, psU, 5mC 10 110.4 Oct4 A, G, psU, 0.5 piC 10 85.0Oct4 A, 0.5 7dG, psU, 0.5 piC 10 58.3 Oct4 A, 0.5 7dG, psU, 5mC 10 27.0Oct4 A, 0.5 7dG, 0.5 5mU, 0.5 piC 20 109.0 Oct4 A, 0.5 7dG, 0.6 5mU, 0.5piC 20 114.8 Oct4 A, 0.5 7dG, 0.7 5mU, 0.5 piC 20 107.2 Oct4 A, 0.5 7dG,0.8 5mU, 0.5 piC 20 110.9 Oct4 A, 0.5 7dG, 0.9 5mU, 0.5 piC 20 103.4Oct4 A, 0.5 7dG, 5mU, 0.5 piC 20 97.8 Oct4 A, 0.5 7dG, psU, 0.5 piC 20124.5 Sox2 A, 0.5 7dG, psU, 0.5 piC 20 109.0 Klf4 A, 0.5 7dG, psU, 0.5piC 20 112.8 c-Myc-2 (T58A) A, 0.5 7dG, psU, 0.5 piC 20 112.8 Lin28 A,0.5 7dG, psU, 0.5 piC 20 126.5 Oct4 A, G, psU, 5mC 20 213.4 Sox2 A, G,psU, 5mC 10 107.2 Klf4 A, G, psU, 5mC 10 106.1 c-Myc-2 (T58A) A, G, psU,5mC 10 97.8 Lin28 A, G, psU, 5mC 10 95.9 Oct4 A, 0.5 7dG, psU, 0.5 piC20 124.2 Sox2 A, 0.5 7dG, psU, 0.5 piC 10 57.3 Klf4 A, 0.5 7dG, psU, 0.5piC 10 59.6 c-Myc-2 (T58A) A, 0.5 7dG, psU, 0.5 piC 10 66.7 Lin28 A, 0.57dG, psU, 0.5 piC 10 65.2 Oct4 A, 0.5 7dG, psU, 0.3 piC 10 60.5 Sox2 A,0.5 7dG, psU, 0.3 piC 10 58.8 Klf4 A, 0.5 7dG, psU, 0.3 piC 10 57.9c-Myc-2 (T58A) A, 0.5 7dG, psU, 0.3 piC 10 62.0 Lin28 A, 0.5 7dG, psU,0.3 piC 10 64.3 Oct4 A, 0.5 7dG, 0.5 5mU, 5mC 10 64.7 Sox2 A, 0.5 7dG,0.5 5mU, 5mC 10 62.4 Klf4 A, 0.5 7dG, 0.5 5mU, 5mC 10 75.6 c-Myc-2(T58A) A, 0.5 7dG, 0.5 5mU, 5mC 10 69.4 Lin28 A, 0.5 7dG, 0.5 5mU, 5mC10 60.7 Oct4 A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC 10 48.3 Sox2 A, 0.5 7dG,0.5 4sU, 0.5 5mU, 5mC 10 54.0 Klf4 A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC 1058.7 c-Myc-2 (T58A) A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC 10 54.7 Lin28 A,0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC 10 54.1 Oct4 A, 0.5 7dG, 0.3 5mU, 5mC 1069.6 Sox2 A, 0.5 7dG, 0.3 5mU, 5mC 10 69.6 Klf4 A, 0.5 7dG, 0.3 5mU, 5mC10 87.4 c-Myc-2 (T58A) A, 0.5 7dG, 0.3 5mU, 5mC 10 68.1 Lin28 A, 0.57dG, 0.3 5mU, 5mC 10 74.3 Oct4 A, 0.5 7dG, 0.4 5mU, 5mC 10 71.3 Sox2 A,0.5 7dG, 0.4 5mU, 5mC 10 69.7 Klf4 A, 0.5 7dG, 0.4 5mU, 5mC 10 74.8c-Myc-2 (T58A) A, 0.5 7dG, 0.4 5mU, 5mC 10 83.7 Lin28 A, 0.5 7dG, 0.45mU, 5mC 10 69.9 XPA-L1 A, G, psU, 5mC 20 120.0 XPA-L2 A, G, psU, 5mC 20114.0 XPA-R1 A, G, psU, 5mC 20 159.6 CCR5-L1 A, G, psU, 5mC 20 170.4CCR5-L2 A, G, psU, 5mC 20 142.8 CCR5-R1 A, G, psU, 5mC 20 132.0 CCR5-R2A, G, psU, 5mC 20 154.8 CCR5-L1 A, G, psU, 5mC 10 56.6 CCR5-L2 A, G,psU, 5mC 10 58.5 CCR5-R1 A, G, psU, 5mC 10 56.8 CCR5-R2 A, G, psU, 5mC10 58.7 TERT-L A, G, U, C 10 49.4 TERT-R A, G, U, C 10 37.6 MYC-L A, G,U, C 10 39.6 MYC-R A, G, U, C 10 33.7 BIRC5-L A, G, U, C 10 63.0 BIRC5-RA, G, U, C 10 44.5 TERT-L A, 0.5 7dG, 0.4 5mU, 5mC 10 50.8 TERT-R A, 0.57dG, 0.4 5mU, 5mC 10 58.3 MYC-L A, 0.5 7dG, 0.4 5mU, 5mC 10 40.8 MYC-RA, 0.5 7dG, 0.4 5mU, 5mC 10 41.4 BIRC5-L A, 0.5 7dG, 0.4 5mU, 5mC 1035.8 BIRC5-R A, 0.5 7dG, 0.4 5mU, 5mC 10 41.5 Oct4 (SEQ ID NO: 8) A, 0.57dG, 0.4 5mU, 5mC 300 2752.0 Sox2 (SEQ ID NO: 9) A, 0.5 7dG, 0.4 5mU,5mC 100 965.0 Klf4 (SEQ ID NO: 10) A, 0.5 7dG, 0.4 5mU, 5mC 100 1093.8c-Myc-2 (T58A) A, 0.5 7dG, 0.4 5mU, 5mC 100 1265.6 Lin28 A, 0.5 7dG, 0.45mU, 5mC 100 1197.8 Oct4 A, 0.5 7dG, 0.35 5mU, 5mC 30 155.7 Sox2 A, 0.57dG, 0.35 5mU, 5mC 15 79.8 Klf4 A, 0.5 7dG, 0.35 5mU, 5mC 15 90.0c-Myc-2 (T58A) A, 0.5 7dG, 0.35 5mU, 5mC 15 83.2 Lin28 A, 0.5 7dG, 0.355mU, 5mC 15 74.0 APP UTR_L (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 20 37.9APPUTR_R (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 20 40.0 APP Exon2L (Rat) A, 0.57dG, 0.4 5mU, 5mC 20 38.6 APP Exon2R (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 2037.9 APP 6L (Human) A, 0.5 7dG, 0.4 5mU, 5mC 20 43.1 APP 6R (Human) A,0.5 7dG, 0.4 5mU, 5mC 20 43.7 APP 7L (Human) A, 0.5 7dG, 0.4 5mU, 5mC 2042.1 APP 7R (Human) A, 0.5 7dG, 0.4 5mU, 5mC 20 36.2 APP 670L (Rat) A,0.5 7dG, 0.4 5mU, 5mC 20 27.0 APP 670R (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 2028.3 APP 678L (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 20 30.1 APP 678R (Rat) A,0.5 7dG, 0.4 5mU, 5mC 20 26.2 APP 680L (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 208.1 APP 680R (Rat) A, 0.5 7dG, 0.4 5mU, 5mC 20 25.4 APP 6L (Human) A,0.5 7dG, 0.4 5mU, 5mC 40 48.6 APP 6R (Human) A, 0.5 7dG, 0.4 5mU, 5mC 4048.6 APP 6L (Human) A, G, U, C 10 54.0 APP 6R (Human) A, G, U, C 10 61.0APP 6L (Human) A, 0.5 7dG, 0.4 5mU, 5mC 10 35.4 APP 6R (Human) A, 0.57dG, 0.4 5mU, 5mC 10 48.0

Example 2 Transfection of Cells with Synthetic RNA

For transfection in 6-well plates, 2 μg RNA and 6 μL transfectionreagent (Lipofectamine RNAiMAX, Life Technologies Corporation) werefirst diluted separately in complexation medium (Opti-MEM, LifeTechnologies Corporation or DMEM/F12+10 μg/mL insulin+5.5 μg/mLtransferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine) to a totalvolume of 60 μL each. Diluted RNA and transfection reagent were thenmixed and incubated for 15 min at room temperature, according to thetransfection reagent-manufacturer's instructions. Complexes were thenadded to cells in culture. Between 30 μL and 240 μL of complexes wereadded to each well of a 6-well plate, which already contained 2 mL oftransfection medium per well. Plates were shaken gently to distributethe complexes throughout the well. Cells were incubated with complexesfor 4 hours to overnight, before replacing the medium with freshtransfection medium (2 mL/well). Volumes were scaled for transfection in24-well and 96-well plates. Alternatively, between 0.5 μg and 5 μg ofRNA and between 2-3 μL of transfection reagent (Lipofectamine 2000, LifeTechnologies Corporation) per μg of RNA were first diluted separately incomplexation medium (Opti-MEM, Life Technologies Corporation orDMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodiumselenite+2 μg/mL ethanolamine) to a total volume of between 5 μL and 100μL each. Diluted RNA and transfection reagent were then mixed andincubated for 10 min at room temperature. Complexes were then added tocells in culture. Between 10 μL and 200 μL of complexes were added toeach well of a 6-well plate, which already contained 2 mL oftransfection medium per well. In certain experiments, DMEM+10% FBS orDMEM+50% FBS was used in place of transfection medium. Plates wereshaken gently to distribute the complexes throughout the well. Cellswere incubated with complexes for 4 hours to overnight. In certainexperiments, the medium was replaced with fresh transfection medium (2mL/well) 4 h or 24 h after transfection.

Example 3 Toxicity of and Protein Translation from Synthetic RNAContaining Non-Canonical Nucleotides

Primary human fibroblasts were transfected according to Example 2, usingRNA synthesized according to Example 1. Cells were fixed and stained20-24 h after transfection using an antibody against Oct4. The relativetoxicity of the RNA was determined by assessing cell density at the timeof fixation.

Example 4 Transfection Medium Formulation

A cell-culture medium was developed to support efficient transfection ofcells with nucleic acids and efficient reprogramming (“transfectionmedium”):

DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+50μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+4 μg/mLcholesterol+1 μM hydrocortisone+25 μg/mL polyoxyethylenesorbitanmonooleate+2 μg/mL D-alpha-tocopherol acetate+20 ng/mL bFGF+5 mg/mLtreated human serum albumin.

A variant of this medium was developed to support robust, long-termculture of a variety of cell types, including pluripotent stem cells(“maintenance medium”):

DMEM/F12+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5 μg/mLtransferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+50 μg/mLL-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+20 ng/mL bFGF+2ng/mL TGF-β1.

Transfection medium, in which the treated human serum albumin wastreated by addition of 32 mM sodium octanoate, followed by heating at60° C. for 4 h, followed by treatment with ion-exchange resin(AG501-X8(D), Bio-Rad Laboratories, Inc.) for 6 h at room temperature,followed by treatment with dextran-coated activated charcoal (C6241,Sigma-Aldrich Co. LLC.) overnight at room temperature, followed bycentrifugation, filtering, adjustment to a 10% solution withnuclease-free water, followed by addition to the other components of themedium, was used as the transfection medium in all Examples describedherein, unless otherwise noted. For reprogramming experiments, cellswere plated either on uncoated plates in DMEM+10%-20% serum or onfibronectin and vitronectin-coated plates in transfection medium, unlessotherwise noted. The transfection medium was not conditioned, unlessotherwise noted. It is recognized that the formulation of thetransfection medium can be adjusted to meet the needs of the specificcell types being cultured. It is further recognized that treated humanserum albumin can be replaced with other treated albumin, for example,treated bovine serum albumin, without negatively affecting theperformance of the medium. It is further recognized that other glutaminesources can be used instead of or in addition to L-alanyl-L-glutamine,for example, L-glutamine, that other buffering systems can be usedinstead of or in addition to HEPES, for example, phosphate, bicarbonate,etc., that selenium can be provided in other forms instead of or inaddition to sodium selenite, for example, selenous acid, that otherantioxidants can be used instead of or in addition to L-ascorbic acid2-phosphate sesquimagnesium salt hydrate and/or D-alpha-tocopherolacetate, for example, L-ascorbic acid, that other surfactants can beused instead of or in addition to polyoxyethylenesorbitan monooleate,for example, Pluronic F-68 and/or Pluronic F-127, that other basal mediacan be used instead of or in addition to DMEM/F12, for example, MEM,DMEM, etc., and that the components of the culture medium can be variedwith time, for example, by using a medium without TGF-β from day 0 today 5, and then using a medium containing 2 ng/mL TGF-β after day 5,without negatively affecting the performance of the medium. It isfurther recognized that other ingredients can be added, for example,fatty acids, lysophosphatidic acid, lysosphingomyelin,sphingosine-1-phosphate, other sphingolipids, ROCK inhibitors, includingY-27632 and thiazovivin, members of the TGF-β/NODAL family of proteins,IL-6, members of the Wnt family of proteins, etc., at appropriateconcentrations, without negatively affecting the performance of themedium, and that ingredients that are known to promote or inhibit thegrowth of specific cell types and/or agonists and/or antagonists ofproteins or other molecules that are known to promote or inhibit thegrowth of specific cell types can be added to the medium at appropriateconcentrations when it is used with those cell types without negativelyaffecting the performance of the medium, for example,sphingosine-1-phosphate and pluripotent stem cells. The presentinvention relates equally to ingredients that are added as purifiedcompounds, to ingredients that are added as parts of well-definedmixtures, to ingredients that are added as parts of complex or undefinedmixtures, for example, animal or plant oils, and to ingredients that areadded by biological processes, for example, conditioning. Theconcentrations of the components can be varied from the listed valueswithin ranges that will be obvious to persons skilled in the art withoutnegatively affecting the performance of the medium. An animalcomponent-free version of the medium was produced by using recombinantversions of all protein ingredients, and non-animal-derived versions ofall other components, including semi-synthetic plant-derived cholesterol(Avanti Polar Lipids, Inc.).

Example 5 Reprogramming Human Fibroblasts Using Synthetic RNA ContainingNon-Canonical Nucleotides

Primary human neonatal fibroblasts were plated in 6-well plates coatedwith recombinant human fibronectin and recombinant human vitronectin(each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, andincubated at room temperature for 1 h) at a density of 10,000 cells/wellin transfection medium. The following day, the cells were transfected asin Example 2, using RNA containing A, 0.5 7 dG, 0.5 5 mU, and 5 mC, andan RNA dose of 0.5 μg/well on day 1, 0.5 μg/well on day 2, 2 μg/well onday 3, 2 μg/well on day 4, and 4 μg/well on day 5. Small colonies ofcells exhibiting morphology consistent with reprogramming became visibleas early as day 5. The medium was replaced with maintenance medium onday 6. Cells were stained using an antibody against Oct4. Oct4-positivecolonies of cells exhibiting a morphology consistent with reprogrammingwere visible throughout the well (FIG. 2).

Example 6 Feeder-Free, Passage-Free, Immunosuppressant-Free,Conditioning-Free Reprogramming of Primary Adult Human Fibroblasts UsingSynthetic RNA

Wells of a 6-well plate were coated with a mixture of recombinant humanfibronectin and recombinant human vitronectin (1 μg/mL in DMEM/F12, 1mL/well) for 1 h at room temperature. Primary adult human fibroblastswere plated in the coated wells in transfection medium at a density of10,000 cells/well. Cells were maintained at 37° C., 5% CO₂, and 5% O₂.Beginning the following day, cells were transfected according to Example2 daily for 5 days with RNA synthesized according to Example 1. Thetotal amount of RNA transfected on each of the 5 days was 0.5 μg, 0.5μg, 2 μg, 2 μg, and 4 μg, respectively. Beginning with the fourthtransfection, the medium was replaced twice a day. On the day followingthe final transfection, the medium was replaced with transfectionmedium, supplemented with 10 μM Y-27632. Compact colonies of cells witha reprogrammed morphology were visible in each transfected well by day 4(FIG. 8).

Example 7 Efficient, Rapid Derivation and Reprogramming of Cells fromAdult Human Skin Biopsy Tissue

A full-thickness dermal punch biopsy was performed on a healthy, 31year-old volunteer, according to an approved protocol. Briefly, an areaof skin on the left, upper arm was anesthetized by topical applicationof 2.5% lidocaine. The field was disinfected with 70% isopropanol, and afull-thickness dermal biopsy was performed using a 1.5 mm-diameterpunch. The tissue was rinsed in phosphate-buffered saline (PBS), wasplaced in a 1.5 mL tube containing 250 μL of TrypLE Select CTS (LifeTechnologies Corporation), and was incubated at 37° C. for 30 min. Thetissue was then transferred to a 1.5 mL tube containing 250 μL ofDMEM/F12-CTS (Life Technologies Corporation)+5 mg/mL collagenase, andwas incubated at 37° C. for 2 h. The epidermis was removed usingforceps, and the tissue was mechanically dissociated. Cells were rinsedtwice in DMEM/F12-CTS. Phlebotomy was also performed on the samevolunteer, and venous blood was collected in Vacutainer SST tubes(Becton, Dickinson and Company). Serum was isolated according to themanufacturer's instructions. Isogenic plating medium was prepared bymixing DMEM/F12-CTS+2 mM L-alanyl-L-glutamine (Sigma-Aldrich Co.LLC.)+20% human serum. Cells from the dermal tissue sample were platedin a fibronectin-coated well of a 6-well plate in isogenic platingmedium. Many cells with a fibroblast morphology attached and began tospread by day 2 (FIG. 3A). Cells were expanded and frozen inSynth-a-Freeze (Life Technologies Corporation).

Cells were passaged into 6-well plates at a density of 5,000 cells/well.The following day, the medium was replaced with transfection medium, andthe cells were transfected as in Example 2, using RNA containing A, 0.57 dG, 0.4 5 mU, and 5 mC, and an RNA dose of 0.5 μg/well on day 1, 0.5μg/well on day 2, 2 μg/well on day 3, 2 μg/well on day 4, and 2 μg/wellon day 5. Certain wells received additional 2 μg/well transfections onday 6 and day 7. In addition, certain wells received 2 ng/mL TGF-β1 fromday 4 onward. The medium was replaced with maintenance medium on day 6.Colonies of cells exhibiting morphology consistent with reprogrammingbecame visible between day 5 and day 10 (FIG. 3B). Colonies grewrapidly, and many exhibited a morphology similar to that of embryonicstem-cell colonies (FIG. 3C). Colonies were picked and plated in wellscoated with recombinant human fibronectin and recombinant humanvitronectin (each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1mL/well, incubated at room temperature for 1 h). Cells grew rapidly, andwere passaged to establish lines.

Example 8 Synthesis of RiboSlice Targeting CCR5

RiboSlice pairs targeting the following sequences: L1:TCATTTTCCATACAGTCAGT, L2: TTTTCCATACAGTCAGTATC, R1:TGACTATCTTTAATGTCTGG, and R2: TATCTTTAATGTCTGGAAAT were synthesizedaccording to Example 1 (FIG. 4A and FIG. 4B). These pairs target 20-bpsites within the human CCR5 gene on the sense (L1 and L2) or antisensestrand (R1 and R2). The following pairs were prepared: L1&R1, L1&R2,L2&R1, and L2&R2.

Example 9 Measurement of CCR5 Gene-Editing Efficiency Using aMismatch-Detecting Nuclease

Primary human fibroblasts were plated in 6-well plates coated withrecombinant human fibronectin and recombinant human vitronectin (eachdiluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, andincubated at room temperature for 1 h) at a density of 10,000 cells/wellin transfection medium. The following day, the cells were transfected asin Example 2 with RNA synthesized according to Example 8. Two days afterthe transfection, genomic DNA was isolated and purified. A region withinthe CCR5 gene was amplified by PCR using the primers F:AGCTAGCAGCAAACCTTCCCTTCA and R: AAGGACAATGTTGTAGGGAGCCCA. 150 ng of theamplified PCR product was hybridized with 150 ng of reference DNA in 10mM Tris-Cl+50 mM KCl+1.5 mM MgCl₂. The hybridized DNA was treated with amismatch-detecting endonuclease (SURVEYOR nuclease, Transgenomic, Inc.)and the resulting products were analyzed by agarose gel electrophoresis(FIG. 4C and FIG. 4D).

Example 10 High-Efficiency Gene Editing by Repeated Transfection withRiboSlice

Primary human fibroblasts were plated as in Example 9. The followingday, the cells were transfected as in Example 2 with RNA synthesizedaccording to Example 8. The following day cells in one of the wells weretransfected a second time. Two days after the second transfection, theefficiency of gene editing was measured as in Example 9 (FIG. 4E).

Example 11 Gene-Editing of CCR5 Using RiboSlice and DNA-Free,Feeder-Free, Immunosuppressant-Free, Conditioning-Free Reprogramming ofHuman Fibroblasts

Primary human fibroblasts were plated as in Example 9. The followingday, the cells were transfected as in Example 2 with RNA synthesizedaccording to Example 8. Approximately 48 h later, the cells werereprogrammed according to Example 5, using RNA synthesized according toExample 1. Large colonies of cells with a morphology characteristic ofreprogramming became visible as in Example 5 (FIG. 4F). Colonies werepicked to establish lines. Cell lines were subjected to directsequencing to confirm successful gene editing (FIG. 4G).

Example 12 Personalized Cell-Replacement Therapy for HIV/AIDS ComprisingGene-Edited Reprogrammed Cells

Patient skin cells are gene-edited and reprogrammed to hematopoieticcells according to the present inventors' previously disclosedinventions (U.S. application Ser. No. 13/465,490, U.S. ProvisionalApplication No. 61/637,570, and U.S. Provisional Application No.61/664,494) and/or Example 11. Cells are then enzymatically releasedfrom the culture vessel, and CD34+/CD90+/Lin− or CD34+/CD49f+/Lin− cellsare isolated. Between about 1×10³ and about 1×10⁵ cells are infused intoa main vein of the patient. Hematopoietic cells home to the bone marrowcavity and engraft.

Example 13 Production of APP-Inactivated Rat Embryonic Stem Cells

Rat embryonic stem cells are plated in 6-well plates at a density of10,000 cells/well in rat stem cell medium. The following day, the cellsare transfected as in Example 2 with 0.5 μg/well of RiboSlicesynthesized according to Example 1 targeting the following sequences: L:TTCTGTGGTAAACTCAACAT and R: TCTGACTCCCATTTTCCATT (0.25 μg L and 0.25 μgR).

Example 14 Production of APP-Knockout Rats Using APP-Inactivated RatEmbryonic Stem Cells

Rat embryonic stem cells are gene-editing according to Example 13 andmicroinjected into rat blastocysts. The microinjected blastocysts arethen transferred to a pseudopregnant female rat.

Example 15 Production of APP-Inactivated Embryos for the Generation ofKnockout Rats

A RiboSlice pair targeting the following sequences: L:TTCTGTGGTAAACTCAACAT and R: TCTGACTCCCATTTTCCATT is synthesizedaccording to Example 1. RiboSlice at a concentration of 5 μg/μL isinjected into the pronucleus or cytoplasm of a 1-cell-stage rat embryo.The embryo is then transferred to a pseudopregnant female rat.

Example 16 Transfection of Cells with Synthetic RNA ContainingNon-Canonical Nucleotides and DNA Encoding a Repair Template

For transfection in 6-well plates, 1 μg RNA encoding gene-editingproteins targeting exon 16 of the human APP gene, 1 μg single-strandedrepair template DNA containing a PstI restriction site that was notpresent in the target cells, and 6 μL transfection reagent(Lipofectamine RNAiMAX, Life Technologies Corporation) were firstdiluted separately in complexation medium (Opti-MEM, Life TechnologiesCorporation) to a total volume of 120 μL. Diluted RNA, repair template,and transfection reagent were then mixed and incubated for 15 min atroom temperature, according to the transfection reagent-manufacturer'sinstructions. Complexes were added to cells in culture. Approximately120 μL of complexes were added to each well of a 6-well plate, whichalready contained 2 mL of transfection medium per well. Plates wereshaken gently to distribute the complexes throughout the well. Cellswere incubated with complexes for 4 hours to overnight, before replacingthe medium with fresh transfection medium (2 mL/well). The next day, themedium was changed to DMEM+10% FBS. Two days after transfection, genomicDNA was isolated and purified. A region within the APP gene wasamplified by PCR, and the amplified product was digested with PstI andanalyzed by gel electrophoresis (FIG. 16).

Example 17 Insertion of a Transgene into Rat Embryonic Stem Cells at aSafe Harbor Location

Rat embryonic stem cells are plated in 6-well plates at a density of10,000 cells/well in rat stem cell medium. The following day, the cellsare transfected as in Example 13 with RiboSlice targeting the followingsequences: L: TATCTTCCAGAAAGACTCCA and R: TTCCCTTCCCCCTTCTTCCC,synthesized according to Example 1, and a repair template containing atransgene flanked by two regions each containing approximately 400 basesof homology to the region surrounding the rat Rosa26 locus.

Example 18 Humanized LRRK2 Rat

Rat embryonic stem cells are plated and transfected as in Example 13with RiboSlice targeting the following sequences: L:TTGAAGGCAAAAATGTCCAC and R: TCTCATGTAGGAGTCCAGGA, synthesized accordingto Example 1. Two days after transfection, the cells are transfectedaccording Example 17, wherein the transgene contains the human LRRK2gene, and, optionally, part or all of the human LRRK2 promoter region.

Example 19 Insertion of a Transgene into Human Fibroblasts at a SafeHarbor Location

Primary human fibroblasts are plated as in Example 9. The following day,the cells are transfected as in Example 2 with RiboSlice targeting thefollowing sequences: L: TTATCTGTCCCCTCCACCCC and R:TTTTCTGTCACCAATCCTGT, synthesized according to Example 1, and a repairtemplate containing a transgene flanked by two regions each containingapproximately 400 bases of homology to the region surrounding the humanAAVS1 locus.

Example 20 Inserting an RNAi Sequence into a Safe Harbor Location

Primary human fibroblasts are plated and transfected according toExample 19, wherein the transgene contains a sequence encoding an shRNA,preceded by the PolIII promoter.

Example 21 Gene Editing of Myc Using RiboSlice

Primary human fibroblasts were plated in 6-well plates at a density of50,000 cells/well in DMEM+10% FBS. Two days later, the medium waschanged to transfection medium. Four hours later, the cells weretransfected as in Example 2 with 1 μg/well of RiboSlice targeting thefollowing sequences: L: TCGGCCGCCGCCAAGCTCGT and R:TGCGCGCAGCCTGGTAGGAG, synthesized according to Example 1. The followingday gene-editing efficiency was measured as in Example 9 using thefollowing primers: F: TAACTCAAGACTGCCTCCCGCTTT and R:AGCCCAAGGTTTCAGAGGTGATGA (FIG. 5).

Example 22 Cancer Therapy Comprising RiboSlice Targeting Myc

HeLa cervical carcinoma cells were plated in 6-well plates at a densityof 50,000 cells/well in folate-free DMEM+2 mM L-alanyl-L-glutamine+10%FBS. The following day, the medium was changed to transfection medium.The following day, the cells were transfected as in Example 21.

Example 23 Gene Editing of BIRC5 Using RiboSlice

Primary human fibroblasts were plated in 6-well plates at a density of50,000 cells/well in DMEM+10% FBS. Two days later, the medium waschanged to transfection medium. Four hours later, the cells weretransfected as in Example 2 with 1 μg/well of RiboSlice targeting thefollowing sequences: L: TTGCCCCCTGCCTGGCAGCC and R:TTCTTGAATGTAGAGATGCG, synthesized according to Example 1. The followingday gene-editing efficiency was measured as in Example 9 using thefollowing primers: F: GCGCCATTAACCGCCAGATTTGAA and R:TGGGAGTTCACAACAACAGGGTCT (FIG. 6).

Example 24 Cancer Therapy Comprising RiboSlice Targeting BIRC5

HeLa cervical carcinoma cells were plated in 6-well plates at a densityof 50,000 cells/well in folate-free DMEM+2 mM L-alanyl-L-glutamine+10%FBS. The following day, the medium was changed to transfection medium.The following day, the cells were transfected as in Example 23 (FIG. 7Aand FIG. 7B).

Example 25 Culture of Cancer-Cell Lines

The cancer cell lines HeLa (cervical carcinoma), MDA-MB-231 (breast),HCT 116 (colon), U87 MG (glioma), and U-251 (glioma) were propagated inculture. Cells were cultured in DMEM+10% FBS or DMEM+50% FBS andmaintained at 37° C., 5% CO₂, and either ambient O₂ or 5% O₂. Cells grewrapidly under all conditions, and were routinely passaged every 2-5 daysusing a solution of trypsin in HBSS.

Example 26 RiboSlice Gene-Editing RNA Design Process and Algorithm

The annotated DNA sequence of the BIRC5 gene was retrieved from NCBIusing the eFetch utility and a python script. The same python script wasused to identify the DNA sequences encoding the protein within each ofthe four exons of the BIRC5 gene. The script then searched thesesequences, and the 40 bases flanking each side, for sequence elementssatisfying the following conditions: (i) one element exists on theprimary strand, the other on the complementary strand, (ii) each elementbegins with a T, and (iii) the elements are separated by no fewer than12 bases and no more than 20 bases. Each element was then assigned ascore representing its likelihood of binding to other elements withinthe human genome using Qblast (NCBI). This score was computed as the sumof the inverse of the nine lowest E-values, excluding the match to thetarget sequence. Pair scores were computed by adding the scores for theindividual elements.

Example 27 Synthesis of RNA Encoding Gene-Editing Proteins (RiboSlice)

RNA encoding gene-editing proteins was designed according to Example 26,and synthesized according to Example 1 (Table 10, FIG. 9). The RNA wasdiluted with nuclease-free water to between 200 ng/μL and 500 ng/μL, andwas stored at 4° C.

TABLE 10 RiboSlice Synthesis Template Reaction ivT (SEQ ID of BindingSite) Nucleotides Volume/μL Yield/μg BIRC5-1.1L A, 0.5 7dG, 0.4 5mU, 5mC20 124.1 (SEQ ID NO: 16) BIRC5-1.1R A, 0.5 7dG, 0.4 5mU, 5mC 20 115.6(SEQ ID NO: 17) BIRC5-1.2L A, 0.5 7dG, 0.4 5mU, 5mC 20 120.3 (SEQ ID NO:18) BIRC5-1.2R A, 0.5 7dG, 0.4 5mU, 5mC 20 121.3 (SEQ ID NO: 19)BIRC5-1.3L A, 0.5 7dG, 0.4 5mU, 5mC 20 120.3 (SEQ ID NO: 20) BIRC5-1.3RA, 0.5 7dG, 0.4 5mU, 5mC 20 113.7 (SEQ ID NO: 21) BIRC5-2.1L A, 0.5 7dG,0.4 5mU, 5mC 20 105.3 (SEQ ID NO: 22) BIRC5-2.1R A, 0.5 7dG, 0.4 5mU,5mC 20 120.3 (SEQ ID NO: 23) BIRC5-2.2L A, 0.5 7dG, 0.4 5mU, 5mC 20101.5 (SEQ ID NO: 24) BIRC5-2.2R A, 0.5 7dG, 0.4 5mU, 5mC 20 111.9 (SEQID NO: 25) BIRC5-3.1L A, 0.5 7dG, 0.4 5mU, 5mC 20 107.2 (SEQ ID NO: 26)BIRC5-3.1R A, 0.5 7dG, 0.4 5mU, 5mC 20 113.7 (SEQ ID NO: 27) BIRC5-2.1LA, 0.5 7dG, 0.35 5mU, 5mC 300 577.9 (SEQ ID NO: 22) BIRC5-2.1R A, 0.57dG, 0.35 5mU, 5mC 300 653.6 (SEQ ID NO: 23)

Example 28 Activity Analysis of RiboSlice Targeting BIRC5

Primary adult human fibroblasts were transfected according to Example 2with 6 RiboSlice pairs targeting BIRC5, designed according to Example26, and synthesized according to Example 27. Two days aftertransfection, genomic DNA was isolated and purified. To measuregene-editing efficiency, 150 ng of the amplified PCR product washybridized with 150 ng of reference DNA in 10 mM Tris-Cl+50 mM KCl+1.5mM MgCl₂. The hybridized DNA was treated with the SURVEYORmismatch-specific endonuclease (Transgenomic, Inc.), and the resultingproducts were analyzed by agarose gel electrophoresis (FIG. 10A). Allsix of the tested RiboSlice pairs efficiently edited the BIRC5 gene, asdemonstrated by the appearance of bands of the expected sizes (asterisksin FIG. 10A).

Example 29 Mitosis-Inhibition Analysis of RiboSlice Targeting BIRC5

Primary adult human fibroblasts were gene edited according to Example28, and were then propagated in culture. After 11 days, genomic DNA wasisolated and purified, and gene-editing efficiency was measured as inExample 28 (FIG. 10B). None of the tested RiboSlice pairs inhibited theproliferation of the fibroblasts, as shown by the appearance of bands ofthe expected sizes (asterisks in FIG. 10B) in genomic DNA isolated fromthe proliferating cells, demonstrating the low toxicity to normalfibroblasts of these RiboSlice pairs.

Example 30 Anti-Cancer-Activity Analysis of RiboSlice Targeting BIRC5

Primary adult human fibroblasts and HeLa cervical carcinoma cells,cultured according to Example 25 were transfected with RiboSlice pairsaccording to Example 28. Proliferation of the fibroblasts slowed brieflydue to transfection reagent-associated toxicity, but recovered within 2days of transfection. In contrast, proliferation of HeLa cells slowedmarkedly, and many enlarged cells with fragmented nuclei were observedin transfected wells. After 2-3 days, many cells exhibited morphologyindicative of apoptosis, demonstrating the potent anti-cancer activityof RiboSlice targeting BIRC5.

Example 31 In Vivo RiboSlice Safety Study

40 female NCr nu/nu mice were injected subcutaneously with 5×10⁶MDA-MB-231 tumor cells in 50% Matrigel (BD Biosciences). Cell injectionvolume was 0.2 mL/mouse. The age of the mice at the start of the studywas 8 to 12 weeks. A pair match was conducted, and animals were dividedinto 4 groups of 10 animals each when the tumors reached an average sizeof 100-150 mm³, and treatment was begun. Body weight was measured everyday for the first 5 days, and then biweekly to the end of the study.Treatment consisted of RiboSlice BIRC5-1.2 complexed with a vehicle(Lipofectamine 2000, Life Technologies Corporation). To prepare thedosing solution for each group, 308 μL of complexation buffer (Opti-MEM,Life Technologies Corporation) was pipetted into each of two sterile,RNase-free 1.5 mL tubes. 22 μL of RiboSlice BIRC5-1.2 (500 ng/μL) wasadded to one of the two tubes, and the contents of the tube were mixedby pipetting. 22 μL of vehicle was added to the second tube. Thecontents of the second tube were mixed, and then transferred to thefirst tube, and mixed with the contents of the first tube by pipettingto form complexes. Complexes were incubated at room temperature for 10min. During the incubation, syringes were loaded Animals were injectedeither intravenously or intratumorally with a total dose of 1 μgRNA/animal in 60 μL total volume/animal. A total of 5 treatments weregiven, with injections performed every other day. Doses were notadjusted for body weight. Animals were followed for 17 days. Nosignificant reduction in mean body weight was observed (FIG. 11;RiboSlice BIRC5-1.2 is labeled “ZK1”), demonstrating the in vivo safetyof RiboSlice gene-editing RNA.

Example 32 Anti-Cancer-Activity Analysis of RiboSlice Targeting BIRC5 ina Glioma Model

The U-251 glioma cell line, cultured according to Example 25, wastransfected with RiboSlice pairs according to Example 28. Glioma cellsresponded to treatment similarly to HeLa cells: proliferation slowedmarkedly, and many enlarged cells with fragmented nuclei were observedin transfected wells. After 2-3 days, many cells exhibited morphologyindicative of apoptosis, demonstrating the potent anti-cancer activityof RiboSlice targeting BIRC5 in a glioma model.

Example 33 Screening of Reagents for Delivery of Nucleic Acids to Cells

Delivery reagents including polyethyleneimine (PEI), various commerciallipid-based transfection reagents, a peptide-based transfection reagent(N-TER, Sigma-Aldrich Co. LLC.), and several lipid-based andsterol-based delivery reagents were screened for transfection efficiencyand toxicity in vitro. Delivery reagents were complexed with RiboSliceBIRC5-1.2, and complexes were delivered to HeLa cells, culturedaccording to Example 25. Toxicity was assessed by analyzing cell density24 h after transfection. Transfection efficiency was assessed byanalyzing morphological changes, as described in Example 30. The testedreagents exhibited a wide range of toxicities and transfectionefficiencies. Reagents containing a higher proportion of ester bondsexhibited lower toxicities than reagents containing a lower proportionof ester bonds or no ester bonds.

Example 34 High-Concentration Liposomal RiboSlice

High-Concentration Liposomal RiboSlice was prepared by mixing 1 μg RNAat 500 ng/μL with 3 μL of complexation medium (Opti-MEM, LifeTechnologies Corporation), and 2.5 μL of transfection reagent(Lipofectamine 2000, Life Technologies Corporation) per μg of RNA with2.5 μL of complexation medium. Diluted RNA and transfection reagent werethen mixed and incubated for 10 min at room temperature to formHigh-Concentration Liposomal RiboSlice. Alternatively, a transfectionreagent containing DOSPA or DOSPER is used.

Example 35 In Vivo RiboSlice Efficacy Study Subcutaneous Glioma Model

40 female NCr nu/nu mice were injected subcutaneously with 1×10⁷ U-251tumor cells. Cell injection volume was 0.2 mL/mouse. The age of the miceat the start of the study was 8 to 12 weeks. A pair match was conducted,and animals were divided into 4 groups of 10 animals each when thetumors reached an average size of 35-50 mm³, and treatment was begun.Body weight was measured every day for the first 5 days, and thenbiweekly to the end of the study. Caliper measurements were madebiweekly, and tumor size was calculated. Treatment consisted ofRiboSlice BIRC5-2.1 complexed with a vehicle (Lipofectamine 2000, LifeTechnologies Corporation). To prepare the dosing solution, 294 μL ofcomplexation buffer (Opti-MEM, Life Technologies Corporation) waspipetted into a tube containing 196 μL of RiboSlice BIRC5-1.2 (500ng/μL), and the contents of the tube were mixed by pipetting. 245 μL ofcomplexation buffer was pipetted into a tube containing 245 μL ofvehicle. The contents of the second tube were mixed, and thentransferred to the first tube, and mixed with the contents of the firsttube by pipetting to form complexes. Complexes were incubated at roomtemperature for 10 min. During the incubation, syringes were loadedAnimals were injected intratumorally with a total dose of either 2 μg or5 μg RNA/animal in either 20 μL or 50 μL total volume/animal. A total of5 treatments were given, with injections performed every other day.Doses were not adjusted for body weight. Animals were followed for 25days.

Example 36 Synthesis of High-Activity/High-Fidelity RiboSlice InVitro-Transcription Template

An in vitro-transcription template encoding a T7 bacteriophageRNA-polymerase promoter, 5′-untranslated region, strong Kozak sequence,TALE N-terminal domain, 18 repeat sequences designed according toExample 26, TALE C-terminal domain, and nuclease domain comprising theStsI sequence (SEQ ID NO: 1), StsI-HA sequence (SEQ ID NO: 2), StsI-HA2sequence (SEQ ID NO: 3), StsI-UHA sequence (SEQ ID NO: 4), StsI-UHA2sequence (SEQ ID NO: 5), StsI-HF sequence (SEQ ID NO: 6) or StsI-HF2sequence (SEQ ID NO: 7) is synthesized using standard cloning andmolecular biology techniques, or alternatively, is synthesized by directchemical synthesis, for example using a gene fragment assembly technique(e.g., gBlocks, Integrated DNA Technologies, Inc.).

Example 37 Synthesis of High-Activity/High-Fidelity RiboSliceGene-Editing RNA

High-Activity RiboSlice and High-Fidelity RiboSlice are synthesizedaccording to Example 27, using in vitro-transcription templatessynthesized according to Example 36.

Example 38 Generation of RiboSlice-Encoding Replication-IncompetentVirus for Treatment of Proteopathy

A nucleotide sequence comprising RiboSlice targeting a DNA sequence thatencodes a plaque-forming protein sequence is incorporated into amammalian expression vector comprising a replication-incompetent viralgenome, and transfected into a packaging cell line to producereplication-incompetent virus. The culture supernatant is collected, andfiltered using a 0.45 μm filter to remove debris.

Example 39 Generation of RiboSlice-Encoding Replication-CompetentOncolytic Virus for Treatment of Cancer

A nucleotide sequence comprising RiboSlice targeting the BIRC5 gene, isincorporated into a mammalian expression vector comprising areplication-competent viral genome, and transfected into a packagingcell line to produce replication-competent virus. The culturesupernatant is collected and filtered, according to Example 38.

Example 40 In Vivo RiboSlice Efficacy Study Orthotopic Glioma Model,Intrathecal Route of Administration

40 female NCr nu/nu mice are injected intracranially with 1×10⁵ U-251tumor cells. Cell injection volume is 0.02 mL/mouse. The age of the miceat the start of the study is 8 to 12 weeks. After 10 days, animals aredivided into 4 groups of 10 animals each, and treatment is begun. Bodyweight is measured every day for the first 5 days, and then biweekly tothe end of the study. Treatment consists of RiboSlice BIRC5-2.1complexed with a vehicle (Lipofectamine 2000, Life TechnologiesCorporation). To prepare the dosing solution, 294 μL of complexationbuffer (Opti-MEM, Life Technologies Corporation) is pipetted into a tubecontaining 196 μL of RiboSlice BIRC5-1.2 (500 ng/μL), and the contentsof the tube are mixed by pipetting. 245 μL of complexation buffer ispipetted into a tube containing 245 μL of vehicle. The contents of thesecond tube are mixed, and then transferred to the first tube, and mixedwith the contents of the first tube by pipetting to form complexes.Complexes are incubated at room temperature for 10 min. During theincubation, syringes are loaded Animals are injected intrarthecally witha total dose of 1-2 μg RNA/animal in 10-20 μL total volume/animal. Atotal of 5 treatments are given, with injections performed every otherday. Doses are not adjusted for body weight. Animals are followed for 60days.

Example 41 Treatment of Glioma with RiboSlice IV Perfusion

A patient with a diagnosis of glioma is administered 1 mg ofHigh-Concentration Liposomal RiboSlice BIRC5-2.1, prepared according toExample 34 by IV infusion over the course of 1 h, 3 times a week for 4weeks. For an initial tumor volume of greater than 500 mm³, the tumor isdebulked surgically and optionally by radiation therapy and/orchemotherapy before RiboSlice treatment is begun. The patient isoptionally administered TNF-α and/or 5-FU using a standard dosingregimen as a combination therapy.

Example 42 Treatment of Glioma with RiboSlice Replication-CompetentOncolytic Virus

A patient is administered 1 mL of replicating virus particles (1000CFU/mL), prepared according to Example 39, by intrathecal orintracranial injection.

Example 43 Treatment of Parkinson's Disease with RiboSlice TargetingSNCA

A patient with a diagnosis of Parkinson's disease is administered 50 μgof RiboSlice targeting the SNCA gene by intrathecal or intracranialinjection.

Example 44 Treatment of Alzheimer's Disease with RiboSlice Targeting APP

A patient with a diagnosis of Alzheimer's disease is administered 50 μgof RiboSlice targeting the APP gene by intrathecal or intracranialinjection.

Example 45 Treatment of Type II Diabetes with RiboSlice Targeting IAPP

A patient with a diagnosis of type II diabetes is administered 5 mg ofRiboSlice targeting the IAPP gene by intravenous, intraperitoneal orintraportal injection.

Example 46 iRiboSlice Personalized Cancer Therapy

A biopsy is taken from a patient with a diagnosis of cancer. Genomic DNAis isolated and purified from the biopsy, and the sequence of the DNA(either the whole-genome sequence, exome sequence or the sequence of oneor more cancer-associated genes) is determined. A RiboSlice pairtargeting the patient's individual cancer sequence (iRiboSlice) isdesigned according to Example 26 and synthesized according to Example27. The patient is administered the personalized iRiboSlice using aroute of administration appropriate for the location and type of cancer.

Example 47 RiboSlice Mixtures for GeneticallyDiverse/Treatment-Resistant Cancer

A patient with a diagnosis of genetically diverse and/ortreatment-resistant cancer is administered a mixture of RiboSlice pairstargeting multiple cancer-associated genes and/or multiple sequences inone or more cancer-associated genes.

Example 48 Mito-RiboSlice for Mitochondrial Disease

A patient with a diagnosis of a mitochondrial disease is administered 2mg of RiboSlice targeting the disease-associated sequence and containinga mitochondrial localization sequence by intramuscular injection.

Example 49 Treatment of Eye Disease with RiboSlice Eye Drops

A patient with a diagnosis of a corneal or conjunctival disease isadministered RiboSlice formulated as a 0.5% isotonic solution.

Example 50 Treatment of Skin Disease with RiboSlice Topical Formulation

A patient with a diagnosis of a skin disease is administered RiboSliceformulated as a 1% topical cream/ointment containing one or morestabilizers that prevent degradation of the RNA.

Example 51 Treatment of Lung or Respiratory Disease with RiboSliceAerosol Formulation

A patient with a diagnosis of a lung or respiratory disease isadministered RiboSlice formulated as a 0.5% aerosol spray.

Example 52 Treatment of Infectious Disease with RiboSlice Targeting aDNA Sequence Present in the Infectious Agent

A patient with a diagnosis of an infectious disease is administeredRiboSlice targeting a sequence present in the specific infectious agentwith which the patient is infected using a route of administrationappropriate to the location and type of infection, and a doseappropriate for the route of administration and severity of theinfection.

Example 53 Gene-Edited Human Zygotes for In Vitro Fertilization

A human germ cell, zygote or early-stage blastocyst is transfected withRiboSlice targeting a gene that encodes a disease-associated mutation ormutation associated with an undesired trait. The genome ischaracterized, and the cell is prepared for in vitro fertilization.

Example 54 Cleavage-Domain Screen for Activity, Fidelity Enhancement ofGene-Editing Proteins

A panel of RiboSlice pairs, each comprising a different cleavage domain,are designed according to Example 26 and synthesized according toExample 27. The activity of the RiboSlice pairs is determined as inExample 28.

Example 55 Gene-Edited Cells for Screening Parkinson's Disease-CausingToxicants

Primary human adult fibroblasts are gene edited according to Example 28using RiboSlice targeting SNCA (Table 11) and repair templates togenerate cells with the SNCA A30P, E46K, and A53T mutations. Cells arereprogrammed and differentiated to dopaminergic neurons. The neurons areused in a high-throughput α-synuclein-aggregation toxicant-screeningassay to identify toxicants that can contribute to Parkinson's disease.

TABLE 11 RiboSlice Pairs for Generation of SNCA A30P, E46K, and A53T.Target Amino Exon Acid Left RiboSlice Binding Site Right RiboSliceBinding Site Spacing 1 A30 TGAGAAAACCAAACAGGGTG TAGAGAACACCCTCTTTTGT 202 E46 TGTTTTTGTAGGCTCCAAAA TACCTGTTGCCACACCATGC 16 2 A53TCCAAAACCAAGGAGGGAGT TAAGCACAATGGAGCTTACC 19

Example 56 Gene-Edited Cells for Screening Cancer-Causing Toxicants

Primary human adult fibroblasts are gene edited according to Example 28using RiboSlice targeting TP53 (Table 12) and repair templates togenerate cells with the TP53 P47S, R72P, and V217M mutations. Cells arereprogrammed and differentiated to hepatocytes. The hepatocytes are usedin a high-throughput in vitro-transformation toxicant-screening assay toidentify toxicants that can contribute to cancer.

TABLE 12 RiboSlicePairs for Generation of TP53 P47S, R72P, and V217M.Target Amino Exon Acid Left RiboSlice Binding Site Right RiboSliceBinding Site Spacing 4 P47 TCCCAAGCAATGGATGATTT TGAACCATTGTTCAATATCG 154 R72 TGAAGCTCCCAGAATGCCAG TAGGAGCTGCTGGTGCAGGG 19 6 V217TGGATGACAGAAACACTTTT TCAGGCGGCTCATAGGGCAC 15

Example 57 Design and Synthesis of RNA Encoding Engineered Gene-EditingProteins (RiboSlice)

RNA encoding gene-editing proteins designed according to Example 26 wassynthesized according to Example 27 (Table 13). Each gene-editingprotein comprised a DNA-binding domain comprising a transcriptionactivator-like (TAL) effector repeat domain comprising 35-36 aminoacid-long repeat sequences, as indicated in Table 13. Sequence IDnumbers are given for the 36 amino acid-long repeat sequences. The label“18” in the template name indicates that the 18^(th) repeat sequence was36 amino acids long. The label “EO” in the template name indicates thatevery other repeat sequence was 36 amino acids long. The amino acidsfollowing the label “18” or “EO” indicate the amino acids at theC-terminus of the 36 amino acid-long repeat sequence(s). The label“StsI” indicates that the nuclease domain contained the StsI cleavagedomain. Templates without the “StsI” label contained the FokI cleavagedomain.

TABLE 13 RiboSlice Encoding Engineered Gene-Editing Proteins. TemplateReaction ivT (SEQ ID of Repeat Sequence) Nucleotides Volume/μL Yield/μgBIRC5-2.1L-18-AHGGG A, 0.5 7dG, 0.4 5mU, 5mC 20 11.9 (SEQ ID NO: 54)BIRC5-2.1R-18-AHGGG A, 0.5 7dG, 0.4 5mU, 5mC 20 11.9 (SEQ ID NO: 54)BIRC5-2.1L-18-AGHGG A, 0.5 7dG, 0.4 5mU, 5mC 20 10.7 (SEQ ID NO: 55)BIRC5-2.1R-18-AGHGG A, 0.5 7dG, 0.4 5mU, 5mC 20 10.9 (SEQ ID NO: 55)BIRC5-2.1L-18-AHGSG A, 0.5 7dG, 0.4 5mU, 5mC 20 11.9 (SEQ ID NO: 56)BIRC5-2.1R-18-AHGSG A, 0.5 7dG, 0.4 5mU, 5mC 20 12.7 (SEQ ID NO: 56)BIRC5-2.1L-18-AHGGG A, 0.5 7dG, 0.4 5mU, 5mC 20 34.5 (SEQ ID NO: 54)BIRC5-2.1R-18-AHGGG A, 0.5 7dG, 0.4 5mU, 5mC 20 34.8 (SEQ ID NO: 54)BIRC5-2.1L-18-AGHGG A, 0.5 7dG, 0.4 5mU, 5mC 20 32.7 (SEQ ID NO: 55)BIRC5-2.1R-18-AGHGG A, 0.5 7dG, 0.4 5mU, 5mC 20 37.4 (SEQ ID NO: 55)BIRC5-2.1L-18-AHGSG A, 0.5 7dG, 0.4 5mU, 5mC 20 31.5 (SEQ ID NO: 56)BIRC5-2.1R-18-AHGSG A, 0.5 7dG, 0.4 5mU, 5mC 20 34.1 (SEQ ID NO: 56)BIRC5-2.1L A, 0.5 7dG, 0.4 5mU, 5mC 20 34.9 BIRC5-2.1R A, 0.5 7dG, 0.45mU, 5mC 20 25.9 BIRC5-2.1L A, 0.5 7dG, 0.4 5mU, 5mC 20 41.5 BIRC5-2.1RA, 0.5 7dG, 0.4 5mU, 5mC 20 38.8 BIRC5-2.1L-StsI A, 0.5 7dG, 0.4 5mU,5mC 20 22.2 BIRC5-2.1R-StsI A, 0.5 7dG, 0.4 5mU, 5mC 20 18.4BIRC5-2.1L-EO-AGHGG A, 0.5 7dG, 0.4 5mU, 5mC 20 21.6 (SEQ ID NO: 55)BIRC5-2.1L A, 0.5 7dG, 0.4 5mU, 5mC 20 17.3 BIRC5-2.1L-StsI A, G, U, C10 71.3 BIRC5-2.1R-StsI A, G, U, C 10 75.1 BIRC5-2.1L-EO-AGHGG A, G, U,C 10 66.4 (SEQ ID NO: 55) BIRC5-2.1R-EO-AGHGG A, G, U, C 10 52.4 (SEQ IDNO: 55)

Example 58 Activity Analysis of RiboSlice Targeting BIRC5

The activity of RiboSlice molecules synthesized according to Example 57was analyzed according to Example 28 (FIG. 12A, FIG. 12B, and FIG. 14).High-efficiency gene editing was observed in cells expressinggene-editing proteins containing one or more 36 amino acid-long repeatsequences. Gene-editing efficiency was highest in cells expressinggene-editing proteins containing one or more repeat sequences containingthe amino-acid sequence: GHGG.

Example 59 In Vivo RiboSlice AAV Safety and Efficacy Study SubcutaneousGlioma Model, Intratumoral Route of Delivery

Animals were set up with tumors comprising U-251 human glioma cellsaccording to Example 35. AAV serotype 2 encoding GFP, BIRC5-2.1LRiboSlice, and BIRC5-2.1R RiboSlice was prepared according to standardtechniques (AAV-2 Helper Free Expression System, Cell Biolabs, Inc.).Viral stocks were stored at 4° C. (short term) or −80° C. (long term)Animals received intratumoral injections of either 160 μL GFP AAV on day1 or 80 μL BIRC5-2.1L RiboSlice AAV+80 μL BIRC5-2.1R RiboSlice AAV onday 1 and day 15. Animals were followed for 25 days. No significantreduction in mean body weight was observed (FIG. 13A), demonstrating thein vivo safety of RiboSlice AAV. Tumor growth was inhibited in theRiboSlice AAV group (FIG. 13B), demonstrating the in vivo efficacy ofRiboSlice AAV.

Example 60 Treatment of Cancer with RiboSlice AAV

A patient is administered 1 mL of RiboSlice AAV virus particles,prepared according to Example 59, by intrathecal or intracranialinjection. Dosing is repeated as necessary. For a patient with aninitial tumor volume of greater than 500 mm³, the tumor is debulkedsurgically and optionally by radiation therapy and/or chemotherapybefore RiboSlice AAV treatment is begun. The patient is optionallyadministered TNF-α and/or 5-FU using a standard dosing regimen as acombination therapy.

Example 61 iRiboSlice AAV Personalized Cancer Therapy

A biopsy is taken from a patient with a diagnosis of cancer. Genomic DNAis isolated and purified from the biopsy, and the sequence of the DNA(either the whole-genome sequence, exome sequence or sequence of one ormore cancer-associated genes) is determined A RiboSlice pair targetingthe patient's individual cancer sequence (iRiboSlice) is designedaccording to Example 26 and synthesized according to Example 59. Thepatient is administered the personalized iRiboSlice AAV using a route ofadministration appropriate for the location and type of cancer.

Example 62 Liposome Formulation and Nucleic-Acid Encapsulation

Liposomes are prepared using the following formulation: 3.2 mg/mLN-(carbonyl-ethoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE),9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol,2 mg/mL ammonium sulfate, and histidine as a buffer. pH is controlledusing sodium hydroxide and isotonicity is maintained using sucrose. Toform liposomes, lipids are mixed in an organic solvent, dried, hydratedwith agitation, and sized by extrusion through a polycarbonate filterwith a mean pore size of 800 nm. Nucleic acids are encapsulated bycombining 10 μg of the liposome formulation per 1 μg of nucleic acid andincubating at room temperature for 5 minutes.

Example 63 Folate-Targeted Liposome Formulation

Liposomes are prepared according to Example 62, except that 0.27 mg/mL1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-5000] (FA-MPEG5000-DSPE) is added to the lipid mixture.

Example 64 Cancer Therapy Comprising Liposomal RiboSlice Targeting BIRC5

Liposomes encapsulating RiboSlice pairs synthesized according to Example23 are prepared according to Example 62 or Example 63. The liposomes areadministered by injection or intravenous infusion, and tumor responseand interferon plasma levels are monitored daily.

Example 65 Cancer Therapy Comprising Liposomal RiboSlice Targeting aCancer-Associated Gene

Liposomes encapsulating RiboSlice targeting a cancer-associated gene,synthesized according to Example 1, are prepared according to Example 62or Example 63. The liposomes are administered by injection orintravenous infusion, and tumor response and interferon plasma levelsare monitored daily.

Example 66 Therapy Comprising Liposomal Protein-Encoding RNA

Liposomes encapsulating synthetic RNA encoding a therapeutic protein,synthesized according to Example 1, are prepared according to Example 62or Example 63. The liposomes are administered by injection orintravenous infusion.

Example 67 Combination Cancer Therapy Comprising RiboSlice TargetingBIRC5 and TNF-α

Patients are administered isolated limb perfusion (ILP) with tumornecrosis factor alpha (TNF-α) and liposomes encapsulating RiboSlicetargeting BIRC5 (see Example 64). Following warming of the limb,liposomes are injected into the arterial line of the extracorporeal ILPcircuit over approximately 5 minutes, and perfusion proceeds for another85 minutes. After 1-2 days, ILP is repeated with TNF-α injected into thearterial line of the extracorporeal ILP circuit over 3-5 minutes andperfusion continues for an additional 60 minutes. Tumor response andinterferon plasma levels are monitored daily.

Example 68 Combination Cancer Therapy Comprising RiboSlice TargetingBIRC5 and Fluorouracil (5-FU)

On day 1 patients receive a 60-minute intravenous infusion of liposomesencapsulating RiboSlice targeting BIRC5 (see Example 64), followed by a46-hour intravenous infusion of 5-FU on days 2 and 3. Tumor response andinterferon plasma levels are monitored daily.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

1.-149. (canceled)
 150. A composition comprising a nucleic acid moleculeencoding a non-naturally occurring fusion protein, comprising anartificial transcription activator-like (TAL) effector repeat domaincomprising one or more repeat units 36 amino acids in length and havingrestriction endonuclease activity, wherein: the repeat domain isengineered for recognition of a predetermined nucleotide sequence ofbetween 1 and 5 bases in length; and the fusion protein recognizes thepredetermined nucleotide sequence.
 151. The composition of claim 150,wherein each of the repeat units differ by no more than seven aminoacids.
 152. The composition of claim 150, wherein: each of the repeatunits comprise the amino acid sequence: LTPXQVVAIAS, where X is selectedfrom E and Q, and the amino acid sequence LTPXQVVAIAS is followed on thecarboxyl terminus by either one or two amino acids that determinerecognition for one of adenine, cytosine, guanine or thymine.
 153. Thecomposition of claim 150, having about 1.5 to about 28.5 repeat units.154. The composition of claim 150, having about 11.5, about 14.5, about17.5 or about 18.5 repeat units.
 155. The composition of claim 150,wherein the predetermined nucleotide sequence is a promoter region. 156.The composition of claim 150, wherein the nucleic acid molecule is asynthetic RNA molecule.
 157. The composition of claim 156, wherein thesynthetic RNA molecule comprises one or more non-canonical nucleotides.158. The composition of claim 157, wherein the non-canonical nucleotideis selected from the group consisting of 5-methyluridine,5-hydroxyuridine, pseudouridine, 5-methylpseudouridine,5-hydroxypseudouridine, 5-methylcytidine, and 5-hydroxycytidine.
 159. Avector comprising the nucleic acid molecule of claim
 150. 160. Thevector of claim 158, wherein the vector is a viral vector.
 161. Thevector of claim 159, wherein the vector comprises one or more of anadenovirus, a retrovirus, a lentivirus, a herpes virus, anadeno-associated virus, and an engineered virus.
 162. The composition ofclaim 150, wherein the fusion protein comprises the catalytic domain ofa protein selected from FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA,StsI-UHA2, StsI-HF, and StsI-UHF.
 163. The composition of claim 162,wherein the fusion protein comprises an amino acid sequence selectedfrom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:
 53. 164. A method formodifying the genome of a cell, comprising contacting the cell with anucleic acid molecule encoding a non-naturally occurring fusion protein,the fusion protein comprising an artificial transcription activator-like(TAL) effector repeat domain comprising one or more repeat units and anendonuclease domain, wherein: at least one of the repeat units is atleast 36 amino acids in length; the repeat domain is engineered forrecognition of a predetermined nucleotide sequence; and the fusionprotein recognizes the predetermined nucleotide sequence and introducesan endonucleolytic cleavage in a nucleic acid of the cell, whereby thegenome of the cell is modified.
 165. The method of claim 164, whereinthe cell is selected from the group consisting of a eukaryotic cell, ananimal cell, a mammalian cell, a human cell, a plant cell, and aprokaryotic cell.
 166. The method of claim 164, wherein the nucleic acidmolecule is a synthetic RNA molecule.
 167. The method of claim 166,wherein the synthetic RNA molecule comprises one or more non-canonicalnucleotides.
 168. The method of claim 167, wherein the non-canonicalnucleotide is selected from the group consisting of 5-methyluridine,5-hydroxyuridine, pseudouridine, 5-methylpseudouridine,5-hydroxypseudouridine, 5-methylcytidine, and 5-hydroxycytidine. 169.The method of claim 164, wherein the genome modification reducesexpression of one or more proteins in the cell.